Use of cells derived from first trimester umbilical cord tissue

ABSTRACT

A method of isolating a pluripotent cell from human umbilical cord is described herein. The method involves collecting a sample of umbilical cord from fetal tissue obtained at less than 13 weeks of gestation, for example a first trimester umbilical cord. The sample is treated to obtain isolated umbilical cord cells, after which the isolated umbilical cord cells are incubated. Cells obtained in this way can be differentiated for use in treating conditions of cell damage, by supplanting the function of a damaged cell in a condition such as spinal cord injury, cardiovascular injury or heart disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent application Ser. No. 12/114,182 filed May 2, 2008; and claims the benefit of priority of U.S. Provisional Patent Application Ser. No. 60/972,022, filed Sep. 13, 2007 which is hereby incorporated by reference.

FIELD

The present disclosure relates generally to human stem cells. More particularly, the present disclosure relates to a method of isolating and expanding stem cells from first trimester umbilical cords, and uses therefor.

BACKGROUND

Stem cells are unspecialized human or animal cells that can produce mature specialized body cells and at the same time replicate themselves. This ability to differentiate into specialized cells has led to much research into their use for treating such fatal diseases and disorders as Parkinson's and Alzheimer's diseases, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis and rheumatoid arthritis. Stem cells are derived from either embryos or adult tissues. Embryonic stem cells are derived from a blastocyst typically containing 200 to 250 cells. Their use has been hampered by the ethical considerations associated with their isolation.

It has also been reported that mesenchymal-like stem cells can be isolated from the perivascular layer of umbilical cords at birth (HUCPVC), or from blood, bone marrow, skin, and other tissues. These postnatal cells have the ability to self-renew and differentiate to all cell types of mesenchymal lineage. Their use, however, has been hampered by the minimal quantities obtained. Furthermore, adult stem cells have significantly restricted differentiation potential, more DNA damage and shorter life spans as compared with pluripotent stem cells derived from fetal tissue.

U.S. Patent Publication 2005/0148074 A1 (Davies et al.) describes a method of isolating progenitor cells from the Wharton's jelly present in human umbilical cord tissue. However, this method requires tissue to be derived from full-term babies.

It is desirable to provide a method for isolating and expanding stem cells from umbilical cord tissue at a stage earlier than full-term.

SUMMARY

It is an object of the present disclosure to obviate or mitigate at least one disadvantage of previous methods for obtaining or using stem cells.

There is described herein a method for isolating and expanding adult stem cells from first trimester umbilical cords. Advantageously, readily available tissue from first trimester umbilical cords is used to yield relatively large amounts of pluripotent cells. According to one aspect there is provided a method of isolating a pluripotent cell from human umbilical, the method comprising: collecting a sample of umbilical cord from fetal tissue obtained at less than 20 weeks of gestation; treating the sample to obtain isolated umbilical cord cells; and incubating the isolated umbilical cord cells.

In an additional aspect, the method can further comprise maintaining the isolated umbilical cord cells, storing (for example, by freezing) the isolated umbilical cord cells, and thawing and restoring the isolated umbilical cord cells to viability is described.

There is also described herein a method of treating a condition of cellular damage, comprising supplanting the function of a damaged cell with a pluripotent cell from a human umbilical cord, wherein the pluripotent cell is obtained by: collecting an umbilical cord from fetal tissue obtained at less than 13 weeks of gestation, by collecting fetal placenta tissue by surgical aspiration; and separating the umbilical cord from the fetal placenta tissue; treating the umbilical cord to obtain isolated umbilical cord cells, by: washing the umbilical cord with PBS, cutting the umbilical cord, comprising an artery and two vessels, into smaller cut pieces, treating the cut pieces with collagenase to obtain isolated umbilical cord cells, and washing the isolated umbilical cord cells with PBS; and incubating the isolated umbilical cord cells, by suspending the isolated umbilical cord cells in a maintenance medium comprising α-MEM, penicillin-streptomycin, and amphotericin; and maintaining the isolated umbilical cord cells under appropriate growth conditions of 37° C., 5% CO₂; wherein prior to cutting the umbilical cord into smaller cut pieces, the umbilical cord is from 0.5-2.0 cm in length and comprises an artery; wherein the isolated umbilical cord cells express one or more transcription factor associated with undifferentiated stem cells; and the transcription factor is OCT-4, SOX-2, or Nanog; and wherein the condition of cellular damage is spinal cord injury, Parkinson's disease, Alzheimer's disease, cardiovascular injury, heart disease, stroke, burn, diabetes, osteoarthritis or rheumatoid arthritis.

Other aspects and features will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will now be described, by way of example only, with reference to the attached Figures.

FIG. 1 shows a proliferation profile.

FIG. 2 is a micrograph at 100× magnification; cells are shown at second passage (A) and fifteenth passage (B).

FIG. 3 is a micrograph; (A) is at 200× magnification, (B) is at 40× magnification.

FIG. 4 is a micrograph showing immunohistochemical staining.

FIG. 5 is a micrograph showing immunocytochemical staining.

FIG. 6 depicts the results from an RT-PCR assay.

FIG. 7 shows EBs in suspension; (A) Magnification 200×; (B) Magnification 400×.

FIG. 8 illustrates immunocytochemistry to identify enzymatically dispersed EBs expression human embryonic germ layers characterization markers.

FIG. 9 shows expression of differentiation markers in embryo body (EB) by RT-PCR.

FIG. 10 shows HUCPVC differentiation to cardiomyocyte-like cells. (A) Magnification 100×; (B) Magnification 40×.

FIG. 11 shows immunocytochemical detection of mesoderm markers on HUCPVC differentiation into cardiomyocytes.

FIG. 12A shows RT-PCR analysis of first-trimester HUCPV cells expression cardiomyocyte marker genes after in vitro differentiation into cardiomyocytes. Differentiated cells express cTnI (lane 2, 416 bp) and alpha-cardiac actin (lane 6, 418 bp).

FIG. 12B shows RT-PCR analysis of first-trimester HUCPV cells expression cardiomyocyte marker genes after in vitro differentiation into cardiomyocytes, wherein differentiated cells express desmin (lane 2, 408 bp) and beta-myosin heavy chain (lane 6, 205 bp).

FIG. 13 shows cell morphology changes after HUCPVC cells were differentiated into nerve-like cells. (A): magnification 40×; (B) and (C): magnification 100×; and (D): magnification 200×.

FIG. 14 shows immunocytochemical detection of ectoderm markers on HUCPVC differentiation into nerve-like cells. (A) MAP-2; (B) MBP; (C) beta-tubulin; and (D) nestin.

FIG. 15 shows morphologic changes observed in pancreatic differentiation stage. In part (A) (Step 2, Enrichment of Nestin Positive Cells) EBs have attached to the tissue culture dish and have differentiated into pancreatic-like cells. Part (B) (Step 3, Differentiation to Insulin-Secreting Pancreatic Islet-like Clusters) shows high density in central pancreatic-like cells.

FIG. 16 shows immunocytochemical staining of HUCPVC-derived islet clusters with pancreatic markers in the presence of (A) insulin; and (B) glucagon.

FIG. 17 shows that HUCPV cells can differentiate into osteogenic and adipogenic lineages. In (A), cells appear polygonal (osteoblasts) under the culture condition of osteogenic differentiation. In (B), cells were stained with Alizarin Red S. In (C), cells were stained with Oil Red O after cultured with adipogenic complete medium.

FIG. 18 shows animal survival, weight and functional readouts over 10 weeks post-SCI.

DETAILED DESCRIPTION

A method is described for isolating and expanding stem cells, and more particularly a method wherein the stem cells are derived from first trimester umbilical cords. In accordance with one aspect described herein, there is provided a method of isolating a pluripotent cell from human umbilical cord, said method comprising: collecting a sample of umbilical cord from fetal tissue obtained at less than 20 weeks of gestation; treating the sample to obtain isolated umbilical cord cells; and incubating the isolated umbilical cord cells. Additional optional steps may also include: maintaining the isolated umbilical cord cells, freezing the isolated umbilical cord cells, and thawing and restoring the isolated umbilical cord cells to viability. In one embodiment, umbilical cord samples can be obtained at less than 13 weeks of gestation.

There is described herein a method of treating a condition of cellular damage. The method comprises supplanting the function of a damaged cell with a pluripotent cell from a human umbilical cord. The pluripotent cell is obtained by: collecting an umbilical cord from fetal tissue obtained at less than 13 weeks of gestation, by collecting fetal placenta tissue by surgical aspiration; and separating the umbilical cord from the fetal placenta tissue; treating the umbilical cord to obtain isolated umbilical cord cells, by: washing the umbilical cord with PBS, cutting the umbilical cord, comprising an artery and two vessels, into smaller cut pieces, treating the cut pieces with collagenase to obtain isolated umbilical cord cells, and washing the isolated umbilical cord cells with PBS; and incubating the isolated umbilical cord cells, by suspending the isolated umbilical cord cells in a maintenance medium comprising α-MEM, penicillin-streptomycin, and amphotericin; and maintaining the isolated umbilical cord cells under appropriate growth conditions of 37° C., 5% CO₂; wherein prior to cutting the umbilical cord into smaller cut pieces, the umbilical cord is from 0.5-2.0 cm in length and comprises an artery; wherein the isolated umbilical cord cells express one or more transcription factor associated with undifferentiated stem cells; and the transcription factor is OCT-4, SOX-2, or Nanog; and wherein the condition of cellular damage is spinal cord injury, Parkinson's disease, Alzheimer's disease, cardiovascular injury, heart disease, stroke, burn, diabetes, osteoarthritis or rheumatoid arthritis.

The method may involve maintaining the isolated umbilical cord cells and changing the maintenance medium every 3-7 days; freezing the isolated umbilical cord cells; and thawing and restoring the isolated umbilical cord cells to viability. Optionally, the collagenase is Type I at 1 mg/mL.

Further, the method may involve maintaining the isolated umbilical cord cells by washing the isolated umbilical cord cells with PBS; adding trypsin-EDTA; harvesting the isolated umbilical cord cells into a tube containing maintenance medium; separating the isolated umbilical cord cells from the maintenance medium; mixing the isolated umbilical cord cells with new maintenance medium; diluting the new maintenance medium containing the isolated umbilical cord cells with additional maintenance medium to obtain a diluted maintenance medium containing the isolated umbilical cord cells; and maintaining the diluted maintenance medium containing the isolated umbilical cord cells under appropriate growth conditions of 37° C., 5% CO₂ and changing the maintenance medium every 3-7 days.

The method may involve freezing the isolated umbilical cord cells by washing the isolated umbilical cord cells with PBS; adding trypsin-EDTA; harvesting the isolated umbilical cord cells into a tube containing maintenance medium; separating the isolated umbilical cord cells from the maintenance medium; cooling the isolated umbilical cord cells to 4° C.; mixing the isolated umbilical cord cells with a freezing medium at 4° C., said freezing medium comprising DMSO; transferring the freezing medium containing the isolated umbilical cord cells to vials pre-chilled to −70° C.; storing the vials at −70° C. for 24 h; and storing the vials in liquid nitrogen.

The method may optionally involve thawing and restoring the isolated umbilical cord cells, which comprises: warming the vials 37° C.; separating the isolated umbilical cord cells from the freezing medium; mixing the isolated umbilical cord cells with maintenance medium; maintaining the maintenance medium containing the isolated umbilical cord cells under appropriate growth conditions of 37° C., 5% CO₂, for 24 hours; replacing the maintenance medium with new maintenance medium; and maintaining the new maintenance medium containing the material under appropriate growth conditions, said conditions being 37° C., 5% CO₂ and changing the new maintenance medium every 3-7 days.

The method described herein may involve maintaining the isolated umbilical cord cells with the following steps: (i) aspirating the medium from the cells when the density of cells exceeds about 70% of the surface of a culture dish or flask in which the cells are maintained, (ii) washing the cells with PBS; (iii) adding trypsin-EDTA to cover the cells until the cells lift off of the dish or flask; and (iv) resuspending the cells in maintenance medium.

The damaged cell according to the method may be supplanted by the pluripotent cell differentiated to a neuronal cell, an osteoblast, a chondrocyte, a myocyte, an adipocyte, or a β-pancreatic islet cell. For example, the pluripotent cell is differentiated to a neuronal cell. This may be in a situation where the condition comprises spinal cord injury, Parkinson's disease or Alzheimer's disease, and spinal cord injury in particular. The pluripotent cell may be differentiated to a cardiomyocyte, which would be the situation when the condition is cardiovascular injury or heart disease.

According to one exemplary embodiment, collection of the sample can comprise: collecting fetal placenta tissue by surgical aspiration; and separating the umbilical cords from the fetal placenta tissue. Furthermore, the step of treating the sample can comprise: washing the umbilical cord with PBS; cutting the umbilical cord into pieces; treating the umbilical cord pieces with collagenase to obtain isolated umbilical cord cells; and washing the isolated umbilical cord cells with PBS. Any type of collagenase may be used provided it achieves the effect of separating the cells such as, for example, Type I collagenase at 1 mg/mL.

The incubating step may comprise: suspending the isolated umbilical cord cells in a maintenance medium composed of α-MEM, Fetal Bovine Serum, penicillin-streptomycin, and amphotericin; and maintaining the material under appropriate growth conditions of 37° C., 5% CO₂ and changing the maintenance medium every 3-7 days. Other maintenance medium or growth conditions may be appropriate as long as cell viability or growth is maintained.

In accordance with one embodiment, the present method can comprise an optional step of maintaining the isolated umbilical cord cells. Ideally, this step can comprise: washing the isolated umbilical cord cells with PBS; adding trypsin-EDTA; harvesting the isolated umbilical cord cells into a tube containing maintenance medium; separating the isolated umbilical cord cells from the maintenance medium; mixing the isolated umbilical cord cells with new maintenance medium; diluting the new maintenance medium containing the isolated umbilical cord cells with additional maintenance medium to obtain a diluted maintenance medium containing the isolated umbilical cord cells; and maintaining the diluted maintenance medium containing the isolated umbilical cord cells under appropriate growth conditions of 37° C., 5% CO₂ and changing the maintenance medium every 3-7 days. Other maintenance medium or growth conditions may be appropriate as long as cell viability or growth is maintained.

As mentioned above, another optional step in the method includes freezing the isolated umbilical cord cells. In one embodiment, this step may comprise: washing the isolated umbilical cord cells with PBS; adding trypsin-EDTA; harvesting the isolated umbilical cord cells into a tube containing maintenance medium; separating the isolated umbilical cord cells from the maintenance medium; cooling the isolated umbilical cord cells to 4° C.; mixing the isolated umbilical cord cells with a freezing medium at 4° C., the freezing medium comprising 80% Fetal Calf Serum and 20% DMSO; transferring the freezing medium containing the isolated umbilical cord cells to vials pre-chilled to −70° C.; storing the vials at −70° C. for 24 h; and storing the vials in liquid nitrogen. Other freezing medium may be appropriate as long as cell viability is maintained.

A further optional step in the described method includes thawing and restoring the isolated umbilical cord cells. In one embodiment, this step may comprise: warming the vials to 37° C.; separating the isolated umbilical cord cells from the freezing medium; mixing the isolated umbilical cord cells with maintenance medium; maintaining the maintenance medium containing the isolated umbilical cord cells under appropriate growth conditions of 37° C., 5% CO₂, for 24 hours; replacing the maintenance medium with new maintenance medium; and maintaining the new maintenance medium containing the material under appropriate growth conditions, said conditions being 37° C., 5% CO₂ and changing the new maintenance medium every 3-7 days. Other maintenance medium or growth conditions may be appropriate as long as cell viability or growth is restored.

The isolated umbilical cord cells may express one or more transcription factors associated with undifferentiated stem cells. Exemplary transcription factors include OCT-4, SOX-2, or Nanog.

The cells isolated according to the method described may undergo transformation into a differentiated cell such as a neuronal cell, an osteoblast, a chondrocyte, a myocyte, an adipocyte, or a β-pancreatic islet cell.

In accordance with another aspect, there is provided a method of obtaining a differentiated cell. In one embodiment, this method involves isolating the cell as described above from human umbilical cord and transforming it using any method acceptable to a person skilled in the art.

Treatment of conditions may be achieved as described herein where the condition requires the function of a damaged cell to be supplanted by a cell obtained according to the method described above. Such conditions may include Parkinson's disease, Alzheimer's disease, spinal cord injury, stroke, burn, heart disease, diabetes, osteoarthritis or rheumatoid arthritis.

Pluripotent cells or differentiated cells can be obtained by methods described herein.

While the first trimester of human gestation can be chronologically identified as the first 13 weeks of gestation, as used herein the term “first trimester” can be extended to further encompass from the 14th week to the 20th week of gestation. A person skilled in the art would recognize that cells isolated up to the 20th week of gestation according to the method described herein may still possess the described features found in those cells isolated in the first 13 weeks of gestation.

The steps described can be further sub-divided. In order to collect samples, fresh fetal-placenta samples are collected from first trimester terminated pregnancies by surgical aspiration into an aseptic bottle; the samples are then moved into dishes where they are searched using forceps, blades and Iris™ scissors for umbilical cords, which are removed.

In one embodiment, treating the umbilical cord sample involves: washing the sample several times with the Dulbecco's Phosphate Buffered Saline (PBS); cutting the sample into small pieces with the curved surgical scissors; transferring the cut sample into a centrifuge tube; digesting the sample; centrifuging; aspirating the supernatant; and washing the sample.

The resulting umbilical cord cells isolated from the treatment of the umbilical cord sample can be incubated by: preparing a warm maintenance medium; re-suspending the isolated umbilical cord cells in the maintenance medium; transferring the isolated umbilical cord cells into tissue culture dishes; adding maintenance medium; and keeping the isolated umbilical cord cells under appropriate growth conditions.

An optional step for the method of isolating cells includes maintaining the isolated umbilical cord cells. The maintenance of isolated umbilical cord cells is undertaken before the cells reach confluence or prior to the growth medium becoming acidic, whichever occurs first. However, the density of cells should exceed approximately 70% of the surface of the culture dishes. In order to maintain the isolated umbilical cord cells, essentially all of the medium is removed from the tissue culture dishes by aspiration; the cells are rinsed once with warmed PBS; room-temperature trypsin-EDTA is added to cover the cells; and the cells are incubated with the trypsin-EDTA until the cells begin to lift off. Cells are harvested into a tube containing maintenance medium and centrifuged to pellet the cell suspension; the media is removed by aspiration and the cells are re-suspended in maintenance medium; and a fraction of the maintenance medium is transferred into a flask containing maintenance medium. Maintenance of the cells in an undifferentiated state can be accomplished by repeating this procedure when the density of the cells exceeds approximately 70% of the surface of the culture flask at each passage.

Another optional step of the method of isolating cells is the storage of the isolated umbilical cord cells. Storage typically involves: freshly preparing freezing medium; harvesting isolated umbilical cord cells using trypsin-EDTA as described above to obtain harvested cells; briefly chilling the harvested cells; re-suspending the harvested cells in ice-cold freezing medium; placing the harvested in pre-chilled cryovials; placing the cryovials in a freezer for 24 hours; and transferring the cryovials to liquid nitrogen for long term storage.

Another optional step of the method of isolating cells is the thawing and restoring of the isolated umbilical cord cells to viability. This step typically involves: warming the maintenance medium; transferring the cryovial in the freezer containing the isolated umbilical cord cells to a water bath; transferring the isolated umbilical cord cells in the cryovial into a centrifuge tube and centrifuging; aspirating the supernatant and re-suspending the isolated umbilical cord cells in an appropriate amount of maintenance medium; and, at a predetermined time after thawing the isolated umbilical cord cells, removing all of the medium and replacing with fresh maintenance medium.

EXAMPLES Example 1

Obtaining First Trimester HUCPV cells

The following method describes obtaining first trimester human umbilical cord perivascular cells (HUPVC).

Materials—Reagents

A non-exhaustive list of possible reagents for use in the described method is provided below. Penicillin-streptomycin liquid containing 5000 U of penicillin and 5000 mg of streptomycin/mL (GIBCO; cat. no. 15070-63), aliquot and store at −20° C. Amphotericin B solution (250 μg/ml, Sigma; cat. no. A-2942), aliquot and store at −20° C. Dulbecco's phosphate-buffered saline (PBS) (+) (GIBCO™; cat. no. 14040-133), store at 4° C. Sterile water, tissue culture grade (GIBCO; cat. no. 15230-162), store at 4° C. Collagenase Type 1 (GIBCO; cat. no. 21985-023), store at 4° C. α-MEM (GIBCO; cat. no. 12571). Defined fetal bovine serum (HyClone, Logan, Utah; cat. no. 30070-03) aliquot and store at −20° C. Trypsin 0.25%/EDTA (GIBCO; cat. no. 25200-056), store at −20° C. DMSO (Sigma™, cat. no. D-5879), store at room temperature. Any additional and acceptable reagents may be used.

Materials—Equipment

A non-exhaustive list of possible equipment to be used in the described method follows. Watchmakers' forceps (Fine Science Tools Inc., Vancouver, Canada); Iris scissors (Fine Science Tools Inc., cat. no. 14060-09) and dissecting curved surgical scissors (Fischer Scientific; cat. no. 08-935); single edge blades; Pipetmen (2, 10, 100, 200, and 1000 μL); 1-mL individually wrapped serological pipet (BD Biosciences; cat. no. 357522); 5-mL individually wrapped serological pipet (BD Biosciences; cat. no. 357543); 10-mL individually wrapped serological pipet (BD Biosciences; cat. no. 357551); 25-mL individually wrapped serological pipet (BD Biosciences; cat. no. 357525); 15 ml conical centrifuge tubes, high-clarity polypropylene (BD Biosciences; cat. no. 352196); 50 ml conical centrifuge tubes, high-clarity polypropylene (BD Biosciences; cat. no. 352070); 100×20 mm tissue culture dishes (TPP, cat no. 93100); 100×15 mm Petri dishes (Sigma, P5731); 75 cm² tissue culture flask (BD Biosciences; cat. no. 354114); Nalgene freezing box (Nalge Nunc, Rochester, N.Y.; cat. no. 5100-0001); Cryogenic vials (VWR; cat. no. CA66008-284); UV tissue culture enclosure hood (Labconco); 37° C. water bath (VWR); Humidified incubator (Fisher); Inverted microscope with a range of phase contrast objectives (X4, X10, X20, and X40) (Zeiss); liquid nitrogen storage tank; and a tabletop centrifuge. Any additional and acceptable equipment may be used.

Collection of Samples

Fresh fetal-placenta samples were collected from pregnancies terminated in the first trimester. The samples were surgically aspirated into an aseptic bottle, and immediately transported from operation room (OR) to the research lab for processing. All further steps described were undertaken in sterile conditions using appropriately sterile techniques. Samples were moved into the 100×15 mm petri dish. The samples were carefully searched for the umbilical cord using the forceps, blades and Iris™ scissors. The first trimester umbilical cord is a clear tube-like tissue connected to placental tissue. It is 0.5-2.0 cm in length and contains 2 vessels and one artery. The rest of any of the samples was put back into the bottle, which was filled with 10% formalin and pathology analysis.

Treatment of the Samples

Isolated umbilical cord was washed several times with PBS. The umbilical cord was cut into small pieces with the curved Iris™ scissors, and transferred into a 15 mL centrifuge tube. The sample was treated for 1 h with collagenase Type 1 (1 mg/ml) while in the 37° C. water bath. The sample was centrifuged at 800 rpm for 15 min at 4° C. to obtain a cell pellet comprising isolated umbilical cord cells and a collagenase supernatant. The collagenase supernatant was removed by aspiration. The cell pellet was washed twice with PBS, centrifuging each time at 800 rpm for 10 min at 4° C. to obtain a washed cell pellet and a PBS supernatant. The PBS supernatant was removed by aspiration.

Incubating the Isolated Umbilical Cord Cells

Maintenance medium (50 ml) was prepared by mixing 44 ml of α-MEM with 5 ml FBS, 0.5 ml of 100× Penicillin-streptomycin aliquot, and 100× 0.5 ml Amphotericin B. The maintenance medium was warmed to 37° C. in the water bath before use. After treating the sample, the isolated umbilical cord cells were re-suspended in 1 mL of maintenance medium to obtain cells. The cells were transferred into 100×20 mm tissue culture dishes and 9 mL of maintenance medium was added. The dishes were placed in the incubator at 37° C. and 5% CO₂ for 3-7 days. The maintenance medium was changed every 2-3 days.

Maintenance of the Isolated Umbilical Cord Cells

The media was aspirated from the culture dishes and the cultures were rinsed once with PBS warmed to 37° C. in a water bath. Sufficient room temperature trypsin-EDTA was added to cover the cells, approximately 4 mL for a 100 mm dish. The cells were allowed to incubate at 37° C. until the cells just began to lift off. The cells were harvested into a tube containing 4 mL of maintenance medium and centrifuged to pellet the cell suspension (800 rpm for approximately 10 minutes). The media was removed by aspiration and the cells were re-suspended in approximately 2 mL of maintenance medium. Pipetting up and down against the bottom of the tube 4-6 times ensured that the cell pellet was disrupted to a single cell suspension.

In order to perform a 1:4 split of the cells, 0.5 mL of cells were transferred into a 75 cm² flask containing the balance of 10 mL of maintenance medium. The remainder of the cells could be stored or used for experiments. Continued maintenance as required to sustain the cells in an undifferentiated state was accomplished by repeat the above procedure when the density of cells exceeded 70% of the surface of the culture flask at each passage.

Storage of Material

Fresh freezing medium was prepared by mixing 80% FCS and 20% DMSO. Cryovials were pre-chilled to −70° C. in a Nalgene™ freezing box and cells at a concentration of between 5×10⁵ and 1×10⁶ cells/mL were harvested with trypsin-EDTA as described above. The majority of the media was removed by aspiration and the cells were chilled on ice for 1-2 min. The final cell pellet was re-suspended in ice-cold freezing medium. The cell solution (0.5 mL per cryovial) was transferred into the pre-chilled cryovials in the Nalgene™ freezing box. The Nalgene™ freezing box containing the cryovials was placed in a −70° C. freezer to arrive at frozen cells. Twenty four (24) hours after the cryovials were placed in the freezer, the frozen cells were transferred to liquid nitrogen for long term storage.

Thawing and Restoring the Material to Viability

Maintenance medium was warmed to 37° C. The cryovial containing the frozen cells was removed from the freezer and thawed quickly in a 37° C. water bath. Once thawed, the cells were transferred into 15 mL conical centrifuge tubes and centrifuged at 800 rpm for 10 minutes. The supernatant was removed by aspiration and the cell pellet was re-suspended in maintenance medium (5 mL for a 60 mm dish or 25 cm² flask; 10 mL for a 100 mm dish). Pipetting gently ensured that the cell pellet was disrupted into a single cell suspension. Twenty four (24) hours after thawing the cells all media was removed and replaced with fresh maintenance medium.

Results

FIG. 1 illustrates the proliferation profile of the cells in the material isolated from first trimester human umbilical cord. The number of cells is plotted against the number of days in culture. The figure shows an increase in cell number over 6 days, indicating that the cells are dividing.

FIG. 2 shows the morphology of the cells in the material isolated from first trimester human umbilical cord. The cells showed homogeneous fibroblast-like morphology. Part A of the figure shows the initial population of the cells at the second passage, while Part B of the figure shows the population of the cells at the 15^(th) passage. In both parts, the magnification is 100×.

FIG. 3 shows the colony-forming ability of the cells in the material isolated from first trimester human umbilical cord. Cells at earlier passages had a higher frequency of colony-formation as shown in the figure. Part A of the figure shows said cells at the third passage (200× magnification), while Part B of the figure shows said cells at the 7^(th) passage (40× magnification).

FIG. 4 shows the immunohistochemical (IHC) detection of early embryonic stem cell markers on the cells of first trimester human umbilical cord. IHC analysis revealed that the umbilical cord tissue was positive for TRA-1-60 (part D), TRA-1-81 (part F), SSEA-3 (part C), SSEA-4 (part E) and Oct-4 (part B), but not SSEA-1 (part A). This positive staining was mainly located in the perivascular cell population. This demonstrates that these cells have embryonic stem cell-like properties and derive mainly from the perivascular cell population. This appears to suggest that the markers are characteristic of embryonic stem cells. However, tumor cells that de-differentiate may express some of these markers. They are markers present on embryonic stem cells indicating that they could have the potential for pluripotential differentiation.

FIG. 5 shows the immunocytochemical detection of early embryonic stem cell markers on the 7^(th) passage of cells in the material isolated from first trimester human umbilical cord. This detection reveals the same markers as those found in the immunohistochemical analysis of the umbilical cord. Expression of these embryonic stem cell markers was consistent from passage 0 to 16, over 11 weeks of culture. Markers: SSEA-1 (part A), OCT-4 (part B), SSEA-3 (part C), SSEA-4 (part D), TRA-1-61 (part E), and TRA-1-80 (part F).

FIG. 6 shows the expression of OCT-4, SOX-2, Nanog and Telemerase transcripts by cells, from passages 1 to 15, in the material isolated from first trimester human umbilical cord. This figure shows that the cells retain expression of these early stem cell markers, and thus retain embryonic stem cell-like properties, for numerous passages in routine culture conditions without showing signs of spontaneous differentiation.

The presence of early embryonic stem markers, along with characteristics such as colony-forming ability, may indicate that material isolated from first trimester umbilical cord cells have embryonic stem cell-like properties. The data provided herein describe immunohistochemical staining that shows the isolated human umbilical cord cells are derived mainly from the perivascular cell population. In addition, the data illustrates the possibility of cryogenic storage and expansion of the isolated human umbilical cord cells, which can be kept undifferentiated in culture. The human umbilical cords isolated from first trimester terminated pregnancies, therefore, have the potential to be a large, and readily obtained source of stem cells. Advantageously, the method described herein does not utilize non-human based feeder layers.

Example 2

Differentiation of First Trimester HUCPV Cells In Vitro and Expression of Tissue-Specific Markers.

Stem cells were isolated and expanded from the first trimester human umbilical cord (HUCPV), demonstrating that the cells have embryonic stem cell characteristics. These cells can express embryonic stem cell markers from passage 0 to 16 and have the ability to self-renew.

First trimester HUCPV cells were differentiated in vitro and examined for the expression of tissue-specific markers in the differentiated cells.

Methods

Perivascular cells were isolated from first trimester umbilical cords and were expanded in α-MEM containing 5-10% of FCS. These cells were grown in a suspension to induce their differentiation into EBs. EBs were transferred onto coated plates and cultured under appropriate condition. Morphology change was examined in these differentiation conditions. Dispersed EBs and differentiated cells were characterized using immunocytochemistry (ICC). RT-PCR assays were used to detect the presence of several tissue-specific molecular markers.

Results

FIG. 7 shows that first trimester HUCPV cells have the ability to form EBs in a suspension culture condition. EBs in suspension may appear individually or as aggregates. (A) shows magnification 200×; and (B) shows magnification 400×.

FIG. 8 shows that EBs can express protein markers characteristic of mesoderm (SMA and cTnI), endoderm (AFP), and ectoderm (nestin, MAP-2). FIG. 8 illustrates immunocytochemistry to identify enzymatically dispersed EBs expression human embryonic germ layers characterization markers.

FIG. 9 shows expression of differentiation markers in embryo body (EB) by RT-PCR. This demonstrates the expression of three germ layer markers: PDX-1, insulin, nestin, cTnI and α-cardiac actin.

FIG. 10 shows that HUCPV cells have morphology change in cardiomyocytes culture condition. (A) shows magnification 100×; and (B) shows magnification 40×.

FIG. 11 shows immunocytochemical detection of mesoderm markers on HUCPVC differentiation into cardiomyocytes. Positive immunostaining was identified for cTnI, actin, and desmin.

FIGS. 9 to 11 show that cell morphology changed under differentiation culture conditions.

FIG. 12A shows RT-PCR analysis of first-trimester HUCPV cells expression cardiomyocyte marker genes after in vitro differentiation into cardiomyocytes. FIG. 12A shows differentiated cells express cTnI (lane 2, 416 bp) and alpha-cardiac actin (lane 6, 418 bp). FIG. 12B shows that differentiated cells express desmin (lane 2, 408 bp) and beta-myosin heavy chain (lane 6, 205 bp).

FIG. 13 shows that nerve-like cells can be observed under neural culture conditions. (A) shows magnification 40×; (B) and (C) show magnification 100×; and (D) shows magnification 200×.

FIG. 14 shows nerve-like cells identified by ICC analysis using neural marker MAP-2, MBP, nestin and β-tubulin. This shows immunocytochemical detection of ectoderm markers on HUCPVC differentiation into nerve-like cells. (A) shows MAP-2; (B) shows MBP; (C) shows beta-tubulin; and (D) shows nestin.

FIG. 15. shows that morphologic changes were observed in pancreatic differentiation stage. In FIG. 15A (Step 2, Enrichment of Nestin Positive Cells), EBs have attached to the tissue culture dish and have differentiated into pancreatic-like cells. In FIG. 15B (Step 3, Differentiation to Insulin-Secreting Pancreatic Islet-like Clusters), high density in central pancreatic-like cells. During this differentiation stage, islets have a three-dimensional topology.

FIG. 16 shows immunocytochemical staining of HUCPVC-derived islet clusters with pancreatic markers. The islet-like clusters can be stained with insulin (A) and glucagon (B).

FIG. 17 shows that HUCPV cells can differentiate into osteogenic and adipogenic lineages. These differentiations can be detected by Alizarin Red S and Oil Red O staining. In (A), cells appear polygonal (osteoblasts) under the culture condition of osteogenic differentiation. In (B), cells were stained with Alizarin Red S. In (C), cells were stained with Oil Red O after cultured with adipogenic complete medium.

The above results show that cells derived from human first trimester umbilical cords represent an embryonic-like stem cell population with the capacity to form EBs in vitro. Further, the results show that the cells also have the capacity to differentiate into a wide variety of cell types that include derivatives of all three embryonic germ layers.

Example 3

Use of Early Intravenous First Trimester Human Umbilical Cord Cells in Spinal Cord Injury

Summary

This Example shows that early intravenous first trimester human umbilical cord-perivascular cell delivery after traumatic spinal cord injury significantly improves forelimb motor function and promotes weight gain. Localized vascular disruption as a result of spinal cord injury (SCI) triggers a cascade of secondary events, including inflammation, reactive gliosis and scarring that influence recovery. In addition to immunomodulatory and pathotropic properties, mesenchymal stromal cells (MSCs), including human umbilical cord matrix cells (HUCMCs), possess pericytic features. This makes MSCs ideally suited to targeting acute vascular disruption, which could reduce the severity of secondary injury, enhance tissue preservation and repair, and promote chronic functional recovery. Here, intravenous tail vein infusion of HUCMCs, first trimester human umbilical cord perivascular cells (FTM-HUCPVCs), adult bone marrow mesenchymal stem cells (BMSCs) and HBSS+2 mM EDTA (HE) control was performed 1 hour post-SCI followed by weekly behavioral testing. Rats were sacrificed 10 weeks post-SCI and immunohistochemistry was employed following perfusion and tissue fixation. Very high resolution ultrasound was used to measure lesional cavitation in live animals. At sacrifice, FTM HUCPVC-infused rats displayed the greatest improvement in grip strength (p<0.05 vs HE). As well, only FTM HUCPVC-infusion led to significant weight gain.

All cell infusion treatments resulted in reduced glial scarring (p<0.05 vs HE). Whilst percent cavitation was reduced with cell infusion, this was not significant. Unlike BMSCs, FTM HUC PVC- and term HUCMC-infusion led to increased axonal (NF200 antibody), myelin (FluoroMyelin stain), and vascular (LEA stain) densities (p<0.05 vs HE). Selective chronic functional recovery was demonstrated alongside histological improvements with acute umbilical cord-derived MSC infusion, most dramatically with FTM-HUCPVCs, in a clinically-pertinent model of cervical SCI. These findings highlight the potential of these cells, isolated according to the method described herein, for early therapeutic intervention in SCI.

In data provided and described herein, it is shown that early intravenous human umbilical cord-derived cell infusion in a clinically relevant model of SCI leads to long-term histological benefits including enhanced vascularisation, myelination and axonal density, reduced glial scarring and cavitation. Of the cells types tested, infusion of FTM-HUCPVCs resulted in the most significant selective functional benefits; specifically improved grip strength and body weight. This is the first study to compare MSCs derived from different donor ages and sources, and the first to identify similarities and differences between their efficacies on several chronic histological and functional parameters after traumatic SCI. Very high resolution ultrasound was used to accurately measure lesional cavitation in live animals. This minimally invasive and effective approach to cell therapy has significant translational implications to the acute treatment of traumatic SCI and other CNS injuries for the purposes of sustained benefit.

Material & Methods

Experimental Design. Passage-matched (passage 6 to 7) FTM HUCPVCs, term HUCMCs, adult BMSCs, new-born fibroblasts cultured under identical conditions were compared as detailed below. Animals were randomly assigned to a treatment group (10 per condition/treatment) and given a coded designation until data acquisition and processing was complete. Rats were sacrificed 4 or 10 weeks post-SCI.

Animal use. All experiments involving animal use were approved by the University Health Network Animal Care Committee (Animal Use Protocol #979), in Toronto, Canada.

Tissue procurement & Cell isolation. Two to three centimeters of term umbilical cord was purchased from LifeLine Stem Cells (New Haven, Ind., USA), and dissected in sterile Hank's Buffered Saline (HBSS, Gibco, Canada) containing 1% gentamycin using aseptic technique. The vein and arteries were carefully removed. Pieces of Wharton's jelly (umbilical cord matrix) were then transferred to collagenase I and II (1 mg/ml, Gibco, Canada), diced into small pieces and incubated for up to 2 hours at 37° C. on an orbital shaker before adding Ca²⁺ and Mg²⁺-free Phosphate-Buffered Saline (PBS, Gibco, Canada) to the viscous cell suspension, followed by trituration, and centrifugation at 2000 rpm for 10 minutes at room temperature.

FTM HUCPVCs were isolated as described herein from Create Fertility Centre (University of Toronto, Toronto, Canada). Other cell types used included passage-matched adult human bone marrow stromal cells (BMSCs, Lonza) and newborn (CRL-2703, ATCC) human fibroblasts. Identical culture conditions were used for all cell types and sources.

Cell culture. Cells were seeded into uncoated tissue culture T175 flasks (Greiner, Canada) in Alpha-MEM (Gibco, Canada) containing 10% batch-tested foetal bovine serum (FBS, HyClone, USA Lot #KTJ32091), 1% sodium pyruvate, 1% Glutamax and 0.1% gentamycin (Sigma-Aldrich, Canada) (complete medium). This culture was referred to as passage 1 (p #1). After 2 to 5 days, the non-adherent HUCMCs were discarded. Fresh complete medium was added to the original dish (p #1) and the adherent cells were grown to a maximum 70-80% confluence before passaging, harvesting for infusion or cryogenic storage. The medium was completely replaced twice a week.

Sub-confluent passage number 6-7 cultures were passaged at no more than 70-80% confluence using trypsin-like enzyme (TLE, Sigma-Aldrich, Canada). The cells were resuspended in 1 ml fresh medium and dissociated by gentle trituration. After passing the cell suspension through a 70 μm filter to remove any clumps, total cell counts and viabilities were determined haemocytometrically using the Trypan blue dye (Sigma-Aldrich, UK) exclusion assay. Cells were reseeded into fresh uncoated 140 mm tissue culture dishes (Greiner, Canada) at 5×10⁵ cells per 20 ml. In order to prevent re-aggregation prior to infusion, cells were instead re-suspended in Hank's buffer containing 2 mM EDTA and were kept on ice for no more than 2 hours after dissociation.

SCI Model and Cell Infusion. All surgeries were performed by the same person (referenced herein as: RV). Young adult female Wistar rats (250-300 g) received a C7 35 g clip compression SCI for one minute under Isoflurane anaesthesia with a 1:1 mixture of O2/NO2 (1 L/min). Then 2.5 million cells were systematically infused in 1 ml Hank's buffer/2 mM EDTA (to prevent re-aggregation prior to infusion) via the tail vein 1 hour post-SCI, which translates into a realistic timeframe of acute intervention of a few hours in a patient. Cells were kept on ice for no more than 2 hours after dissociation. Infusion via the tail vein was performed manually but over a duration of no less than 5 minutes. Increasing the cell number and/or infusion any sooner was associated with a higher rate of mortality occurring immediately after cell infusion. Specialised post-operative husbandry protocols were followed by qualified and experienced personnel (also blinded to treatment conditions) to maximise animal welfare. These included corn cob (1¼″) and Isopad™ bedding, Clavamox-supplemented drinking water and Bacon Softies diet (starting immediately postop). Animals were caged singly post-operatively with Nylabone™ and kept on a 12-hour light/dark cycle at all times. Bladder squeezing (three times a day), cyclosporine (Sandimmune) injections (10 mg/kg s.c. once daily) and Buprenorphine injections (0.05 mg/kg s.c. twice daily) were performed at regular times.

Careful consideration was given to approved standards of reporting for all procedures and data described.

Lesional Spinal Cord Cavitation & Neuroanatomical Assessment with Very High Resolution Ultrasound (VHRUS) and Unbiased Cavalieri Estimation. The injured spinal cord was assessed by pre-sacrificial echography with very high-resolution ultrasound (VHRUS) with a 44 MHz probe (Vevo 770, VisualSonics, Toronto, Canada). Three-dimensional (3D) VHRUS acquisitions were made in B-mode. The 3D files were analysed with ImageJ software with minor modifications. Instead of using a set region of interest (ROI), the bright pixel lesions were delineated by three independent blinded observers within a 15 sagittal image slice, 102 μm thick stack to generate a reproducible cavity volume.

Histology. Spinal cords were isolated from rats perfused with 250 ml chilled PBS (without Ca²⁺ and Mg²⁺) followed by 50 ml of chilled 4% paraformaldehyde (PFA). Spinal cords were then post-fixed in 4% PFA overnight at 4° C. before transfer to 30% sucrose (at 4° C.) and storage in OCT (at 4° C. for 1-2 days then −20° C.) until embedding in fresh OCT and cryosectioning (20 μm thick cross-sections). The delay between spinal cord isolation and cryosectioning was minimised as much as possible. To assess vascularity, endothelial cells were labelled with a DyLight 594-conjugated tomato lectin from Lycopersicon esculentum agglutinin (LEA, VectorLabs DL-1177, 1:300). Myelination and axonal density were quantified using FluoroMyelin (Molecular Probes F34651; 1:100) and anti-NF200 (Sigma N0142, 1:200), respectively. Gliosis and glial scarring were quantified using anti-GFAP (Millipore AB5541, 1:200) and anti-CSPG (Clone CS-56, Sigma C8035, 1:200) antibodies, respectively. All appropriate goat secondary antibodies (Alexa Fluor) were used at a 1:200 dilution.

Slides were baked for 15 minutes at 55° C. so cryosections would adhere permanently to the slides. After blocking in PBS (without Ca²⁺ and Mg²⁺)+2% FBS+0.1% Triton-X for 1 hour at room temperature, primary antibodies diluted in the same diluent solution were applied overnight at 4° C. After 3 washes in PBS, secondary antibodies (1:200 dilution) were applied as necessary. Hoechst 33242 was used as nuclear counterstain. Slides were mounted in Mowiol after 5 PBS washes. Negative staining controls (primary or secondary antibody alone) were used to assess baseline image acquisition parameters. The latter were kept consistent for each fluorescent channel between slides and treatment conditions. All staining was done in one batch and the delay between mounting slides and image acquisition was kept to a minimum.

Unbiased Cavalieri Estimation of cavitation, tissue sparing and grey:white matter ratio was carried out on StereoInvestigator software on a Nikon Eclipse E800 microscope on longitudinal cryosections slides.

Image Acquisition & Processing Protocol. Images were acquired at ×200 magnification. From 3 sections per rat, various view fields spanning a minimum of 5 mm rostrocaudal to the injury site were stitched automatically post-acquisition by the StereoInvestigator software on a Nikon Eclipse E800 microscope. Images were then thresholded (based on negative control slides) and binarised, and the area of fluorescent staining was determined as a proportion of the total area (kept fixed) of the lesional and peri-lesional spinal cord corresponding to 5 mm of tissue. Any cavity was excluded during this measurement. Cavitation was expressed as a proportion of the total volume of spinal cord corresponding to 6 mm of lesional and peri-lesional tissue, which equates to the acquisition viewfield of VHRUS.

Statistical Analyses. Statistical analyses were performed with GraphPad Prism software (La Jolla, Calif., USA). Each test is described in the corresponding figure legend. Unless otherwise stated, One-Way ANOVAs and Bonferroni's multiple comparisons test were performed, with the Alpha threshold set to 0.05. Standard errors of means were compared using the Brown-Forsythe test.

Results

Functional Readouts. The antigenic profiles of the cells employed in this study were evaluated. A battery of standard weekly functional tests starting one week post-SCI (FIG. 18). All animals started off within the same weight range (250-270 g) at week 0. No animals showed any overt signs of infection that might impede weight gain. Rats treated with FTM HUCPVCs exclusively gained weight more than rats receiving other cells acutely and were at a higher pre-sacrificial weight than rats receiving other treatments. The significant weight gain demonstrated by the term HUCMC- and FTM HUCPVC-infused rats over HE-infused controls was not reflected in other functional readouts at the same time points, except for grip strength.

FIG. 18 shows animal survival, weight and functional readouts over 10 weeks post-SCI (1 minute C7 compression). Multiple t-tests with Sidak-Bonferroni correction (HE n=14, Term HUCMCs n=11, FTM HUC PVCs n=8, BMSCs n=8, *p<0.05).

The grip strengths of FTM HUC PVC-infused rats were significantly greater than term HUCMCs from 5 to 10 weeks post-SCI. The two grey asterisks at 2 and 4 weeks post-SCI represent term HUCMCs vs HE p=0.0504 and 0.0514 respectively. Rats treated with FTM HUCPVCs exclusively performed significantly better from 3 weeks post-SCI onwards compared to rats receiving HE vehicle acutely.

No significant differences in inclined plane (combined right and left) were detected between conditions at 1-8 weeks post-SCI, but term HUCMC-infused rats performed better than HE-infused controls at 9 and 10 weeks post-SCI (term HUCMC vs HE p=0.0394 and 0.0042, respectively) and all cell-infused rats were better than HE controls at 10 weeks post-SCI (BMSC vs HE p=0.009, FTM HUCPVC vs HE p=0.0012).

No significant differences were detected between conditions and most animals levelled off at a BBB score (combined right and left) of 9-11.

Chronic Histology

Chronic Tissue Preservation. Next, tissue preservation was examined, specifically: cystic cavitation, tissue sparing, grey:white matter ratio and spinal cord diameter were evaluated. Cavitation was assessed by two complementary techniques—VHRUS immediately prior to sacrifice at 10 weeks and Cavalieri estimation on StereoInvestigator software after histological processing. These two techniques showed a statistically significant correlation (p=0.0472). Although trends of reduced cavitation with cell infusion were clear, no statistically significant effect of cell infusion was found on cavity volume relative to HE controls (term HUCMCs and BMSCs, p=0.5507 and 0.5558 vs HE, respectively). There was also no significant difference in percent cavitation between cell-infused rats. A similar trend of reduced percent cavitation with cell infusion (relative to HE-infused controls) was found when cavity volume was assessed post-sacrificially using unbiased stereological Cavalieri estimation. No other neuroanatomical parameter (tissue sparing, grey:white matter ratio and spinal cord diameter) indicated that tissue preservation was enhanced with cell infusion and no significant difference between conditions was found. The functional benefits of term HUCMC and FTM HUCPVC infusion were not reflected by higher grey:white matter ratio, tissue sparing or lesion site spinal cord diameter compared to controls or other cells.

Cavitation was assessed by Cavalieri estimation on StereoInvestigator software (after histological processing). VHRUS and Cavalieri estimation of cavitation showed a statistically significant correlation (data not shown here). Tissue sparing parameters were assessed by Cavalieri estimation on StereoInvestigator software (after histological processing. As expected, tissue sparing and spinal cord diameter (at the lesion site) showed the inverse trend to % cavitation. Stitch of Longitudinal section (20 μm thick) of Peri-Lesional Spinal Cord 10 weeks post-SCI taken at ×20 was evaluated at ×4 magnification. Slides were stained with LEA (endothelial cells/vasculature), FluoroMyelin or anti-NF200 (axons). All parameters were expressed as the proportion of the area within a 5 mm length of lesioned spinal cord (2.5 mm rostrocaudal to the lesion site) with positive staining.

Vascular density, Myelination & Axonal density. Following sacrifice and isolation of the spinal cord, cryosections were stained for antigenic markers of vascular density (LEA), myelination (FluoroMyelin, FM) and axonal density (NF200). All parameters were expressed as the proportion of the area within a 5 mm length of lesioned spinal cord (2.5 mm rostrocaudal to the lesion site) with positive staining. Only term HUCMC and FTM HUCPVC infusion resulted in significantly higher vascularity, myelination and axonal density compared to HE-infused control rats at 10 weeks post-SCI. There was no significant difference between term HUCMC- and FTM HUCPVC-infused rats for any of these three histological parameters. Adult human BMSC infusion consistently resulted in higher values for these parameters than HE controls without reaching statistical significance. There was no significant difference between HE- and term HUCMC-infused rats at 4 weeks post-SCI for these markers, the only two conditions compared at this time point (data not shown). LEA (endothelial cells/vasculature), FluoroMyelin or anti-NF200 (axons) staining was evaluated at 4 weeks post-SCI. All parameters were expressed as the proportion of the area within a 5 mm length of lesioned spinal cord (2.5 mm rostrocaudal to the lesion site) with positive staining. p values were obtained through the Mann-Whitney test.

Glial Scarring. Antigenic markers of gliosis (GFAP) and glial scarring (CSPG) were examined. Both parameters were expressed as the proportion of the area within a 5 mm length of lesioned spinal cord (2.5 mm rostrocaudal to the lesion site) with positive staining. Infusion of all three cells resulted in significantly lower glial scarring compared to HE-infused control rats. There was no significant difference between term HUCMC-, FTM HUCPVC- and BMSC-infused rats (data not shown). There was no significant difference between HE- and term HUCMC-infused rats at 4 weeks post-SCI for these markers. Stitch of Longitudinal section (20 μm thick) of Peri-Lesional Spinal Cord was evaluated 10 weeks post-SCI taken at ×20 Slides were stained with anti-GFAP (astrocytes/gliosis) and anti-CSPG (glial scar). All parameters were expressed as the proportion of the area within a 5 mm length of lesioned spinal cord (2.5 mm rostrocaudal to the lesion site) with positive staining.

Bladder Size. Bladder size has been correlated with functional performance. Despite the higher than control grip strength and body weight in term HUCMC- and FTM HUCPVC-infused rats, bladder size in this study was no different from controls and rats receiving other cells. Bladder sizes at 10 weeks post-SCI. No significant difference was found between conditions.

Discussion

In this Example, only rats infused with FTM HUCPVCs gained weight significantly more than controls and those receiving other cells over 10 weeks post-SCI. This was also reflected in better grip strength performance after FTM HUCPVC infusion from 2 to 10 weeks post SCI, but not other functional readouts. Inclined plane performance was slightly improved relative to HE controls only at 10 weeks post-SCI and only in rats receiving cell infusions. BBB scores levelled off at 9-10 by 5 weeks post-SCI and no difference was found between conditions. It was found that early systemic infusion of human umbilical cord MSCs, but not adult human BMSCs, significantly promotes vascular density, myelination and axonal density. It was also found that glial scarring is significantly reduced irrespective of which cells are infused. Other neuroanatomical parameters (cystic cavitation, tissue sparing, grey:white matter ratio and spinal cord diameter) and bladder size at 10 weeks post-SCI were not influenced by cell infusion.

The improved vascular density, myelination and axonal density could be a consequence of acute tissue preservation or enhanced repair, or both. There might be a threshold that needs to be achieved, alongside a sufficient reduction of glial scarring in order for functional benefits to become apparent. The absence of infused cells at or near the lesion site (data not shown) suggests the infused cells achieve their benefit through the provision of trophic support

The most effective cell administration route for the integration of donor cells within and near the lesion site is still debated, even though the invasiveness of intra-parenchymal cell injection is well recognised, as well as the challenge this poses to the application of cell therapy especially during the acute phase of injury. However, the benefits of intravenous cell administration are clear from this Example.

Although not yet a validated therapy for SCI, the safety of this approach has already been established through pre-clinical SCI studies, and its clinical application to other conditions affecting the CNS, including multiple sclerosis and bone marrow transplantations. Nevertheless, this approach is not without its limitations. Osteoblastic differentiation of infused MSCs has been reported within lung tumours and although this has not been observed in injury and degeneration models, this possibility should not be dismissed.

At 10 weeks post-SCI, two out of 15 (one sub-cutaneous) of the rats receiving term HUCMCs and two out of 10 rats receiving FTM HUCPVCs intravenously developed tumours located either between the stomach and liver or sub-cutaneously (located on or very near the incision line). Immunostaining for the anti-human nuclear antigen revealed that none of these tumours were of donor (human) origin. Nevertheless, their presence indicates the possibility of the raised risk of tumour development which might be associated with term HUCMC and FTM HUCPVC infusions. In contrast, infusion of term and FTM HUCPVCs in nude mice observed over a 6 month period (n=3) in a myocardial infarction model, revealed no detectable tumor formation (data not shown). Nevertheless, this risk requires further investigation prior to clinical translation.

CONCLUSIONS

The lack of detection of infused cells within the peri-lesional area of the spinal cord imposes limits on the characterisation of the sub-population that reaches and/or survives that is likely to be responsible for localised vasculogenic effects detected. It also suggests that early benefit is preserved chronically even in the absence of infused cells at or near the lesion site. The secretory profiles of the cells used in this study can be used to identify potential molecular mediators of benefit. Culture conditions could be adjusted to prime cells prior to infusion to secrete more pro-angiogenic factors.

The choice of cells is important even though no differences in their acute beneficial effects on the vasculature can be determined. Umbilical cord-derived cells are better than adult BMSCs for chronic neuroanatomical integrity, and are better for glial scar reduction. Other characteristics of these cells may have a role to play in chronic functional recovery from traumatic SCI, including their myogenic differentiation.

This Example shows that the minimally invasive approach of early MSC infusion in a clinically relevant model of moderately severe cervical SCI leads to modest chronic neuroanatomical functional improvements, most prominently by MSC-like cells derived from the first trimester umbilical cord perivascular cells. This suggests that this cellular treatment could be applied in a combinatorial strategy with other approaches to achieve better recovery. This could also be applicable to injuries and neurodegeneration affecting other parts of the CNS.

Example 4

Early Mesenchymal Cell Infusion Reduces Acute Vascular Disruption After Traumatic Spinal Cord Injury

Summary

In this Example, it is demonstrated that early mesenchymal cell infusion reduces acute vascular disruption after traumatic spinal cord injury. Disruption of the blood-spinal cord barrier occurs immediately after spinal cord injury (SCI) and plays a major role in triggering the secondary injury cascade. Mesenchymal Stromal Cells (MSCs), including human umbilical cord matrix cells (HUCMCs), have pericytic attributes and may promote vascular repair, reduce tissue loss and functional impairment. HUCMCs, adult bone marrow MSCs (BMSCs), first trimester human umbilical cord perivascular cells (FTM HUCPVCs) or newborn fibroblasts were infused via the tail vein 1 hour post clip compression-induced cervical SCI in rats, which were sacrificed 1 or 3 days later. Snap-frozen homogenates of spinal cord tissue were used for spectrophotometric quantification of vascular permeability and lesional hemorrhage using Drabkin's assay. Pre-sacrifice, very high frequency ultrasound (VHRUS) measurements of the hemorrhage were undertaken. Vascular density was assessed histologically using LEA staining. It was found that HUCMC- (p=0.038 vs HE), BMSC- and FTM HUCPVC-infused animals had significantly less vascular permeability and parenchymal hemorrhage than HE-(HBSS+2 mM EDTA) or newborn fibroblast-infused rats (p=0.47 vs HE) at 24 hours post-SCI. Similar results were obtained when comparing lesion volumes (VHRUS) at 24 and 72 hours post-SCI. LEA staining indicated that both FTM HUCPVCs and term HUCMC-infusion post-SCI led to significant preservation of blood vessels in the lesioned spinal cord parenchyma 72 hours post-SCI (p=0.0177 vs HE).

The reduction of acute vascular permeability and parenchymal hemorrhage by MSC infusion is demonstrated herein, in a clinically relevant model of cervical SCI. This minimally invasive and effective approach holds translational promise for the acute treatment of traumatic SCI and other CNS injuries.

INTRODUCTION

The prevalence of spinal cord injury (SCI) in Canada and the US exceeds 1.3 million individuals (www.christopherreeve.org), with approximately 13,000 new injuries annually, about 60% of which are in the cervical region. Despite medical advances, many patients with SCI still experience substantial and permanent loss of motor, sensory and autonomic function. As current treatments for SCI are only minimally effective, the development of novel non-invasive strategies to reduce the deleterious effects of secondary pathophysiological events is essential, particularly since it has been shown that a modest improvement in neuroanatomical integrity after SCI can have substantial, clinically relevant impact on neurological recovery.

SCI involves a primary insult, followed by a series of secondary peri-lesional events which include: vascular disruption, cell death, inflammation, ischemia, reactive oxygen species generation, disruption of ion channels, demyelination and reactive gliosis. Some of the reactive efforts intended to limit the extent of damage, paradoxically, exacerbate the condition by inhibiting endogenous regeneration as well as interventional therapeutic treatments by creating an inhibitory milieu within and around the lesion site.

Vascular disruption of the blood-spinal cord barrier (BSCB) and edema play a role in triggering the cascade of secondary events culminating in progressive tissue loss and functional deficits. The BSCB remains compromised long after the initial primary mechanical damage to local vasculature due to the effects of inflammatory mediators (including matrix metalloproteinases) on endothelial cells and loss of astrocytes. Recent studies have shown that vascular stabilization after traumatic SCI can limit the severity of secondary. Limited endogenous neovascularisation is believed to constrain endogenous repair as well as the efficacy of therapeutic interventions. Molecular strategies to promote angiogenesis might support repair but must be balanced against the fact that newly formed blood vessels are more permeable than more mature ones and might exacerbate inflammation. Nevertheless, VEGF, erythropoietin (EPO), FGF1, FGF2, Ang-1, PDGF and statins have all shown promise in pre-clinical studies. However, this purely pharmacological approach cannot address the requirement for precise and dynamic modulation of the peri-lesional concentrations of pro-angiogenic factors.

Cell therapy is well suited to addressing the multi-factorial nature of SCI. Different sources and types of stem/progenitor cells, including embryonic stem cells, neural progenitor cells and bone marrow mesenchymal stem cells (BMSCs) are contemplated. Cell therapy for SCI must overcome the hostile and inhibitory microenvironment of the lesion site to enable implanted cells to integrate within host tissue, or persist long enough to induce paracrine effects, and provide functional benefit. In this regard, mesenchymal stem cells are ideal. Intravenously administered adult BMSCs have been shown to provide benefit in models of SCI. Even with low engraftment, they have been shown to increase the density of new blood vessels in the lesioned spinal cord, which can have functional benefit. Similar observations have been made in models of cerebral ischemia. However, the optimal choice of cells for this mode of treatment remains unknown, despite several clinical trials investigating systemic infusion of bone marrow cells for treatment of SCI.

Typically, adult progenitor cells fare less well than their fetal and embryonic counterparts, not only in terms of proliferation and differentiation, but also with regard to post-implantation survival, migration and integration within the recipient CNS. It was therefore hypothesized that recently characterized cells with MSC-like properties from human umbilical cord tissue would be superior alternative source of MSCs to adult BMSCs.

The umbilical cord consists of an outer layer of amniotic epithelial cells enclosing a gelatinous matrix known as Wharton's jelly, which contains two arteries and a vein. The matrix, especially the perivascular region, is a rich source of progenitor cells. Both first trimester human umbilical cord perivascular cells (FTM HUCPVCs) and term birth human umbilical cord matrix cells (HUCMCs) are similar to BMSCs, but these cells are less mature and have superior proliferation potential, have higher levels of telomerase activity and, unlike BMSCs, they can undergo repeated freeze-thaw cycles without significant loss of viability and without accumulating karyotypic abnormalities. HUCMCs and FTM HUCPVCs are thought to be non-immunogenic, and may even suppress the immune response, potentially making them suitable for allogeneic transplantation. They are highly pathotropic following transplantation, and possibly more so than adult BMSCs. Of greatest relevance to SCI, they secrete a wide range of trophic factors known to promote neural cell survival. HUCMCs have also proven beneficial post-SCI in the clinic.

Acute vascular permeability and parenchymal hemorrhage are reduced by MSC infusion in a clinically relevant model of moderately severe cervical SCI. Vascular disruption is assessed herein in live animals using Very High Resolution Ultrasound, which has been previously validated, and complemented this method with more traditional techniques (Evans blue and Drabkin's assays). This minimally invasive and effective approach to cell therapy has profound translational implications to the acute treatment of traumatic SCI and other CNS injuries.

Material & Methods

Passage-matched (passage 6 to 7) FTM HUCPVCs, term HUCMCs, adult BMSCs, newborn and adult dermal fibroblasts cultured under identical conditions were compared as detailed below. Cells were infused intravenously 1 hour after SCI. Various readouts were obtained 24 or 72 hours later. Experimental rats were randomly divided into 5 per condition/treatment and sacrificed 24 or 72 hours post-SCI. All experiments involving animal use were approved by the University Health Network Animal Care Committee, Toronto Canada.

Cells were infused intravenously 1 hour after SCI. Various readouts were obtained 24 or 72 hours later.

Tissue procurement & Cell isolation. 2-3 cm of term umbilical cord was purchased from LifeLine Stem Cells (New Haven, Ind., USA), and dissected in sterile Hank's Buffered Saline (HBSS, Gibco, Canada) containing 1% gentamycin using aseptic technique. The vein and arteries were carefully removed. Pieces of Wharton's jelly (umbilical cord matrix) were then transferred to collagenase I and II (1 mg/ml, Gibco, Canada), diced into small pieces and incubated for up to 2 hours at 37° C. on an orbital shaker before adding Ca2+ and Mg2+-free Phosphate-Buffered Saline (PBS, Gibco, Canada) to the viscous cell suspension, triturating and centrifuging at 2000 rpm for 10 minutes at room temperature.

FTM HUCPVCs were isolated as described herein, and obtained from Create Fertility Centre (University of Toronto, Canada). Other cells types used included passage-matched adult human bone marrow stromal cells (BMSCs, Lonza), and adult (PCS-201-012, ATCC) and newborn (CRL-2703, ATCC) human fibroblasts. Identical culture conditions were used for all cell types and sources.

Cell culture. Cells were seeded into uncoated tissue culture T175 flasks (Greiner, Canada) in Alpha-MEM (Gibco, Canada) containing 10% batch-tested fetal bovine serum (FBS, HyClone, USA Lot #KTJ32091), 1% sodium pyruvate, 1% Glutamax and 0.1% gentamycin (Sigma-Aldrich, Canada) (complete medium). This culture was referred to as passage 1 (p #1). After 2 to 5 days, the non-adherent HUCMCs were either discarded or reseeded into a fresh dish (p #1 B). Fresh complete medium was added to the original dish (p #1A) and the adherent cells were grown to a maximum 70-80% confluence before passaging, harvesting for infusion or cryogenic storage. The medium was completely replaced twice a week.

Sub-confluent passage number 6-7 cultures were passaged at no more than 70-80% confluence using trypsin-like enzyme (TLE, Sigma-Aldrich, Canada). The cells were resuspended in 1 ml fresh medium and dissociated by gentle trituration. After passing the cell suspension through a 70 μm filter to remove any clumps, total cell counts and viabilities were determined haemocytometrically using the Trypan blue dye (Sigma-Aldrich, UK) exclusion assay. Cells were reseeded into fresh uncoated 140 mm tissue culture dishes (Greiner, Canada) at 5×105 cells per 20 ml. In order to prevent re-aggregation prior to infusion, cells were instead resuspended in Hank's buffer containing 2 mM EDTA and were kept on ice for no more than 2 hours after dissociation.

SCI Model and Cell Infusion. All surgeries were performed by a single researcher. Young adult female Wistar rats (250-300 g) received a C6 28 g or C7 35 g clip compression SCI for 1 minute under Isoflurane anaesthesia with a 1:1 mixture of O₂/NO₂ (1 L/min). Acute vascular disruption post-SCI was targeted by systemically infusing 2.5 million cells in 1 ml Hank's buffer/2 mM EDTA (HE, to prevent re-aggregation prior to infusion) via the tail vein 1 hour post-SCI (translating into a realistic timeframe of acute intervention of a few hours in a human patient). Cells were kept on ice for no more than 2 hours after dissociation. Infusion via the tail vein was performed manually but over a duration of no less than 5 minutes. Increasing the cell number and/or infusion any sooner was associated with a higher rate of mortality occurring immediately after cell infusion. Animals were randomly assigned to a treatment group and given a coded designation until data acquisition and processing was complete. Specialised post-operative husbandry protocols were followed by qualified and experienced personnel (also blinded to treatment conditions) to maximise animal welfare. These included corn cob (1¼″) and Isopad bedding, Clavamox-supplemented drinking water and Bacon Softies diet (starting immediately postop). Animals were caged singly post-operatively with Nylabone and kept on a 12-hour light/dark cycle at all times. Bladder squeezing (three times a day), cyclosporine (Sandimmune) injections (10 mg/kg s.c. once daily) and Buprenorphine injections (0.05 mg/kg s.c. twice daily) were performed at regular times.

The effects of treatment relative to controls were evaluated by measuring hemorrhage, vascular integrity and inflammation. Rats were terminally sedated with Isoflurane and sacrificed by intracardial perfusion with PBS. The spinal cord was isolated along with other organs. A defined length (5 mm) of the lesioned spinal cord was isolated and snap-frozen on dry ice. An equal length of the spinal cord rostral and caudal to the lesioned spinal cord was also isolated and processed identically. Snap-frozen homogenates of 5 mm lesional and 5 mm peri-lesional (rostrocaudal) spinal cord tissue were divided into 3 equal portions for spectrophotometric quantification of Evans blue (EB) dye, myeloperoxidase (MPO) activity assay and Drabkin's assay.

Careful consideration was given to approved standards of reporting for all procedures and data described (Animals in Research: Reporting in Vivo Experiments, ARRIVE and Minimum Information about a Spinal Cord Injury Experiment, MIASCI).

Very High Resolution Ultrasound Imaging (VHRUS) & 3D Lesion Assessment. The injured spinal cord was examined in live rats by echography with very high-resolution ultrasound (VHRUS) with a 44 MHz probe (Vevo 770, VisualSonics, Toronto, Canada). Three-dimensional (3D) VHRUS acquisitions were made in B-mode. The 3D files were analysed with ImageJ software as previously described with minor modifications. Instead of using a set ROI, the bright pixel lesions were delineated by two independent blinded observers within a 15 sagittal image slice stack (102 μm) to generate a reproducible 3D lesion volume.

Parenchymal Hemorrhage—Drabkin's Assay. Parenchymal hemorrhage was assessed via a modified version of the manufacturer's instructions (Sigma D5941). The sample was sonicated in 100 μL of deionized distilled H₂O and subsequently centrifuged at 13000 rpm for 15 minutes. The supernatant was collected and added to complete Drabkin's Reagent (Drabkin's Reagent powder in 1000 mL of distilled H₂O and 0.5 mL of 30% Brij 35 Solution) and allowed to stand for 15 minutes for the reaction to take place (haemoglobin to cyanomethaemoglobin). Colorimetric measurements were performed using the Perkin Elmer Victor2™ spectrophotometer (Wallac 1420 Victor2™, Perkin Elmer, Waltham, USA) at 540 nm, normalized to tissue weight (in grams) and calculated based on a bovine blood haemoglobin (H2500, Sigma-Aldrich, Canada) standard curve.

Vascular permeability—Evans Blue Assay. Wistar rats were infused with 2% Evans blue (EB) in PBS without Ca2+ and Mg2+ 30 minutes prior to sacrificial perfusion with 250 ml PBS without Ca2+ and Mg2+. The third of 5 mm lesion and peri-lesional (rostrocaudal) spinal cord tissue was homogenized in 500 μL of dimethylformamide (DMF) and incubated at 50° C. for 24 hours. The samples were subsequently centrifuged at 13000 rpm for 15 minutes. The supernatant was collected and 150 μl was aliquoted into a 96 well flat bottom glass plate (Zeisser, Germany) and colorimetric measurements were performed using the Perkin Elmer Victor2™ 1420 spectrophotometer at 620 nm. Samples were normalized to tissue weight (g) and EB concentration was calculated based on a standard curve in DMF.

Histology. Spinal cords were isolated from rats perfused with 250 ml chilled PBS (without Calcium and Magnesium) followed by 50 ml of chilled 4% paraformaldehyde (PFA). Spinal cords were then post-fixed in 4% PFA overnight at 4 oC before transfer to 30% sucrose (at 4° C.) and storage in OCT (at 4° C. for 1-2 days then −20° C.) until embedding in fresh OCT and cryosectioning (20 μm thick cross-sections). The delay between spinal cord isolation and cryosectioning was minimised as much as possible. To label endothelial cells, a DyLight 594-conjugated tomato lectin from Lycopersicon esculentum agglutinin (LEA, VectorLabs, 1:300) was used. Anti-human nuclear antigen antibody was used to detect human cells (Millipore MAB1281, 1:200). Slides were baked for 15 minutes at 55° C. so cryosections would adhere permanently to the slides. After blocking in PBS (without Calcium and Magnesium)+2% FBS+0.1% Triton-X for 1 hour at room temperature, primary antibodies diluted in the same diluent solution were applied overnight at 4° C. After 3 washes in PBS, secondary antibodies (1:200 dilution) were applied as necessary. Hoechst 33242 was used as nuclear counterstain. Slides were mounted in Mowiol medium after 5 PBS washes. Negative staining controls (primary or secondary antibody alone) were used to assess baseline image acquisition parameters. The latter were kept consistent for each fluorescent channel between slides and treatment conditions. All staining was performed in one batch and the delay between mounting slides and image acquisition was kept to a minimum. To use as additional positive staining controls, rats were injected intra-spinally with HUCMCs (25 000 cells/3 ul) after C6, 23 g SCI into 4 sites rostrocaudal to the lesion.

Auto-fluorescence Correction Protocol. Hemorrhage interferes with fluorescent microscopy due to the high auto-fluorescence of blood-borne components. Removing this signal manually is not possible. Images (18200×25000 pixels) were segregated into red channel (LEA) and green channel folders, and renamed to matching sample names. The images were then batch-downscaled in ImageJ® to 25% of their original dimensions for feasibility of analysis (Process>Batch>Macro). The red and green images were then thresholded using the Color Threshold function. The images were then saved as <SampleName>-LEA.tif, and using the OR function in the Image Calculator, the overlapping area (corresponding to the auto-fluorescent signal) between images of different channels was determined and saved as <SampleName>-Auto.tif. Following this, the images that were previously generated (<SampleName>-LEA, -Auto.tif) were all opened, and using the SUBTRACT function in the Image Calculator, the computed auto-fluorescence was subtracted from the thresholded LEA images, respectively. The final results were saved as <SampleName>-LEA-Final.tif, and their signal levels were represented as a % area fraction using the Measure function.

Statistical Analyses. Statistical analyses were performed with GraphPad Prism software (La Jolla, Calif., USA). Each test is described in the corresponding figure legend. Unless otherwise stated, One-Way ANOVAs and Bonferroni's multiple comparisons tests were performed. Standard errors of means were compared using the Brown-Forsythe test.

Results

The antigenic profiles of the FTMHUCPVC cells employed in this study have been previously characterised. The majority of HUCMCs expressed the classical mesenchymal markers, including CD90, CD44, CD166, CD105, CD73, prolyl-4-hydroxylase, vimentin, collagen I, alkaline phosphatase but no haematopoietic markers (CD45 and CD34). Most fibroblasts expressed prolyl-4-hydroxylase, collagen I and the proliferative marker Ki67 but not the other MSC and haemotopoietic lineage markers tested. Passage-matched term HUCMCs, FTM-PVCs and BMSCs were morphologically similar (spindle-shaped) and proliferative.

Very High Resolution Ultrasound (VHRUS) enables the visualization of the spinal cord in live animals after laminectomy under anesthesia. The lesion site was examined using this technique after an extra rostral (C6) laminectomy—a double C7-T1 laminectomy had previously been performed for inducing SCI. A significant reduction in parenchymal hemorrhagic lesion volume was found at 24 and 72 hours after 35 g C7 SCI with 1-hour post-SCI infusions of FTM-PVCs, HUCMCs or BMSCs, but not newborn fibroblasts. This difference was no longer detected 1 week post-SCI. There was no statistical difference between the MSC sources.

Haemorrhagic lesion volumes were measured by VHRUS at 24 and 72 hours and 1 week post-C7, 35 g (1 minute) SCI.

Parenchymal hemorrhage was measured after C7, 35 g 1-minute clip compression SCI using the Drabkin's assay. MSC infusion 1 hour after C7 SCI significantly reduced the amount of hemoglobin in the lesioned spinal cord parenchyma 24 hours later, unlike newborn fibroblasts. However, this effect was lost at 72 hours post-SCI. Although a trend for reduced parenchymal hemorrhage was observed rostrocaudal to the lesion at 24 hours following cell infusion relative to vehicle-infused rats (also reduced lesional hemorrhage at 72 hours post-SCI), this failed to achieve statistical significance. No significant difference was seen between the MSC sources.

In order to confirm that these vasoprotective effects were indeed due to live cell infusion and not simply a passive mechanical effect on the spinal cord vasculature (and given the lack of significant difference between MSC sources on vascular permeability), lysed cells were infused. However, these resulted in immediate mortality upon infusion, even after resuspending the lysate in fresh vehicle. The same was seen after adult fibroblast infusion. Therefore term HUCMCs were infused, which had been fixed in 4% paraformaldehyde for 10 minutes, washed and resuspended in 1 ml vehicle per 2.5 million cells. No mortality was observed, but interestingly, parenchymal hemorrhage was reduced at a similar level to that seen with live cells.

Vascular permeability was examined after C7, 35 g 1-minute clip compression SCI using pre-sacrificial EB infusion. MSC infusion 1 hour post-C7 SCI significantly reduced the amount of dye that extravasated into the lesioned spinal cord parenchyma 24 hours later, unlike newborn fibroblasts. This effect was not seen rostrocaudal to the injury and was lost at 72 hours post-SCI at the lesion site. No significant difference was seen between the MSC sources.

In order to identify whether the observations made were specific to C7 injury, the efficacy of vascular permeability reduction with intravenous cell infusion was examined at different clinically relevant injury levels and severities The same significant reduction in vascular permeability was seen at 24 hours post-SCI after HUCMC infusion 1 hour post-SCI at multiple cervical SCI levels and severities but, although the trend was maintained, significance was not reached at T11, 35 g SCI (p=0.2126). The reduction in lesional vascular permeability was smaller after C6, 18 g SCI compared to C6, 28 g but very similar between the two C7 SCI severities tested (28 g and 35 g). Lesional vascular permeability after intravenous term HUCMC infusion at different injury levels and severities was evaluated, with n=5 per group.

The effects of lesional vascular permeability of FTM-PVC, HUCMC and newborn fibroblast infusion 1 hour post-C6 SCI, another clinically relevant model was investigated. A significant reduction in the amount of dye that extravasated into the lesioned spinal cord parenchyma 24 hours post-SCI was found after MSC infusion, but not newborn fibroblasts. Unlike observations made after C7 SCI, this effect was maintained at 72 hours post-C6 SCI. No significant difference was seen between these two umbilical cell sources. This was found upon observing lesional vascular permeability after intravenous cell infusion at 24 and 72 hours after C6, 28 g SCI (n=5 per group).

In order to determine the mechanism of action of infused cells, term HUCMCs were infused intravenously 1 hour post-C6 28 g SCI, Evans blue was injected systemically and rats were sacrificed 30 minutes later. There was no significant reduction of lesional vascular permeability seen at this time point with this treatment. Lesional vascular permeability was assessed at 30 minutes after intravenous term HUCMC infusion 1 hour post-C6, 28 g SCI (n=4 HE; n=6 HUCMC; Unpaired two-tailed T-test with Welch's correction).

Vascularity was examined after C7, 35 g 1-minute clip compression SCI using LEA staining on histological cryosections. LEA labels endothelial cells. Parenchymal hemorrhage was clearly visible in cryosections. After C7 SCI, term HUCMC-infusion 1 hour post-SCI led to a significant preservation of blood vessels in the lesioned spinal cord parenchyma 72 hours post-SCI, unlike newborn fibroblasts. This effect was not seen at 24 hours post-SCI at the lesion site (data not shown). Human cells were not detected after intravenous infusion, even though the antibodies used were validated.

Cross-sections (20 μm thick) of peri-lesional spinal cord were observed at 24 hours post-SCI (×4 magnification). Lesional vascular density represented was assessed by area of staining for LEA (n=3 per condition). As positive staining controls, rats were injected rostrocaudal to the injury site with 90 000 term HUCMCs after a C6, 23 g 1-minute SCI (4 injection sites, 2 rostral and 2 caudal to lesion site; 3 μl per site) at 4 weeks post-SCI and sacrificed 3 days later. 20 μm thick longitudinal sections were stained with anti-human nuclear antigen antibody (Millipore MAB1281, 1:200).

It was found that an unavoidable distortion of the anatomy of the spinal cord occurred during isolation. In addition, many smaller hemorrhagic foci seen during cryosectioning were no longer visible after collection of cryosections onto microscope slides.

VHRUS image showed a loss of morphology during spinal cord isolation, when assessed pre-isolation, after cutting ends of spinal cord; rostral to injury site; and caudal to injury site.

Discussion

This Example found that early systemic infusion of human MSCs of various derivations, not including fibroblasts, significantly reduces acute vascular disruption after traumatic SCI. Vascular disruption was quantified using 3 complementary techniques. VHRUS signal of a hemorrhagic lesion in live animals correlates with both Evans blue-assessed vascular permeability and Drabkin's reagent-assessed parenchymal hemorrhage at 24 hours post-SCI, and Evans blue and Drabkin's assay data correlate with each other at the same time point. Combined VHRUS, Evans blue and Drabkin's quantification of vascular disruption may be more accurate than histological methods because of distortions in the anatomy of the spinal cord during isolation.

Although acute localised vascular disruption occurring post-SCI can trigger and determine the severity of secondary injury and functional impairment, treatment options are limited. Vaso-protective and/or pro-angiogenic cell therapies optimised by comparing multiple human cell types and sources specifically targeting this disruption have not been described for SCI. Previous studies using systemically infused rodent adult MSCs have found reduced acute vascular permeability and improved chronic functional outcome relative to controls. The optimal choice of cells for this mode of treatment remains unknown, despite several clinical trials testing the systemic infusion of bone marrow cells for the treatment of SCI. Although not yet a fully validated therapy for SCI, the safety of this approach has already been established by its clinical application to other conditions, including multiple sclerosis and also in pre-clinical studies.

Despite the effects of reducing vascular disruption, infused cells could not be found within the spinal cord using immunohistological methods. It is assumed that many accumulate in the lungs, liver and spleen and from there (if they survive) are able to secrete trophic and immunomodulatory molecules into the circulation to achieve their observed effects. The fact that no cells could be detected histologically suggests that very few localize or remain in the spinal cord after infusion. This might explain the absence of a detectable effect on peri-lesional neutrophil and macrophage/microglial infiltration. The absence of infused cells within the spinal cord might also be explained by the possibility that they are washed away during the perfusion of the animal prior to isolating the spinal cord. Not perfusing would mean that RBCs/haemoglobin would remain in the tissues and interfere with immunohistochemistry and possibly even with PCR.

The infusion of fixed cells on parenchymal hemorrhage 24 hours post-SCI demonstrates that the hemostatic effects seen in this study rely, not on live infused cells, but rather on a passive mechanical effect likely localized to the peri-lesional site. On the other hand, vascular permeability is likely to rely on live infused cells rather than being a passive mechanical effect since vascular permeability was not significantly reduced 30 minutes after live cell infusion, which would have likely been the case in a purely mechanical hemostatic scenario.

Neuro- and vasoprotective strategies have failed in clinical trials despite promising pre-clinical studies. One reason for this is likely to be the heterogeneity of injuries seen in the clinic as opposed to the standardised models employed and strived for in the laboratory. To address this issue, the efficacy of the approach taken herein was examined by administering cells after SCIs of multiple severities and at multiple levels. From these investigations, it emerged that early systemic cell infusion is effective at reducing BSCB disruption after cervical (which affects the majority of sufferers) but not lower thoracic SCI. Given that neuronal cell bodies have a higher metabolic requirement than axons and glia, gray matter is more highly vascularized than white matter. After SCI in the monkey, gray matter vascular perfusion drops whilst white matter perfusion increases, indicating that ischemic mechanisms play a lesser role in white matter damage following traumatic injury. This is a possible explanation for the greater vulnerability of gray matter (more abundant in cervical levels of the spinal cord) compared to white matter. The lack of significant vascular permeability reduction after T11 SCI compared to cervical SCIs might be a result of the drop in blood pressure after the latter but not the former. This fall in vascular perfusion post-SCI would exacerbate the death and dysfunction of peri-lesional endothelial cells and pericytes.

In this example infused cells could not be detected within the spinal cord even with significant cell infusion-induced reductions in parenchymal vascular permeability.

CONCLUSIONS

For the purpose of acute vasoprotection after SCI, the source of MSCs does not seem to matter but the type of cell used does. The choice of MSC source is determined by practical, ethical and logistical considerations, including ease of isolation and proliferation potential in culture.

Evans blue dye binds circulating albumin to generate a 69 kDa molecule that permeates the disrupted vasculature. However, many cytokines and neurotrophic factors are in the 12-45 kDa size range and might be able to cross the BSCB even when Evans blue-albumin can no longer do so. Thus, the use of smaller molecular markers of vascular disruption alongside Evans blue would be informative. Furthermore, red blood cells are in the 6-8 μm size range. Labelling these extravasating RBCs would also be useful in examining the extent of severe BSCB disruption in future studies.

In an effort to better understand the mechanisms of action of intravenously infused cells on the lesional and peri-lesional vasculature, acute changes in the molecular components of the BSCB can be examined after cell infusion.

Reduction of acute vascular permeability and parenchymal hemorrhage by MSC infusion is shown herein, in a clinically relevant model of moderately severe cervical SCI. This minimally invasive approach to acutely administered cell therapy has the potential to facilitate the clinical deployment of MSCs to reduce the vascular disruption resulting from traumatic CNS injury. As for other traumatic injuries to the CNS, it is unlikely that any single therapeutic approach will treat the myriad of secondary events occurring as a result of the insult. This paradigm is more likely to become part of the arsenal of multi-modal treatments not only for SCI, but also for injuries and neurodegenerative conditions affecting other parts of the CNS, and possibly other organ systems.

Example 5

First Trimester Human Umbilical Cord-Derived Perivascular Cells have Cardiovascular Regenerative Potential

Summary

In this Example, it is shown that first trimester human umbilical cord-derived perivascular cells (FTM HUCPVCs) exhibit superior cardiovascular regenerative potential compared to older sources of human MSCs. Younger human mesenchymal stromal cell (hMSCs) sources can be more effective than older sources of hMSCs for cell-based therapy of myocardial infarction (MI). In this Example, the objective was to compare the ability of first trimester (FTM) HUCPVCs versus other hMSC sources to 1: migrate towards and reconnect injured cardiomyocytes in vitro 2: support angiogenesis 3: engraft in a rat MI model and 4: improve cardiac function after MI. Matrigel™-coated transwell membrane infiltration by FTM, term HUCPVCs or bone marrow stem cells (BMSC) in response to injury of rat cardiomyocytes by wounding or hypoxia, was measured using live fluorophores. Intracellular and mitochondrial dye transfer between hMSCs and cardiomyocytes in co-culture was measured. The effect of hMSCs on developing endothelial networks was evaluated in a modified rat aortic ring assay. hMSCs were injected into the myocardium of Foxmu rats 1 week following coronary artery ligation. At 2 weeks, cardiac function was assessed in rats by echocardiography, cell retention by Alull-based qPCR, and FISH. Scar tissue size and vasculature were assessed by IHC. It was found that FTM HUCPVCs showed increased invasion towards injured cardiomyocytes, reconnected cardiomyocytes, and promoted increased growth of endothelial networks in vitro compared to other hMSCs. Echocardiography showed significantly increased cardiac function in FTM HUCPVC—but not BMSC—and term HUCPVC-treated rats when compared to controls. A 2-fold and 5-fold increase in FTM HUCPVC retention was observed when compared to term HUCPVCs and BMSCs, respectively. FTM HUCPVCs significantly improved scar tissue vasculature. The effect sustained up to 6 weeks in nude mice.

INTRODUCTION

Cardiovascular disease is the leading cause of mortality and morbidity globally. While congenital heart disease affects the young and the middle aged, coronary heart disease predominantly afflicts adults and the elderly. Congestive heart failure (CHF) is the terminal form of all heart diseases, with the highest mortality rates worldwide. In 2015, approximately 5 million people lived with CHF in the USA.

During an episode of acute myocardial infarction (MI), impaired blood flow due to an obstructed coronary artery triggers an ischemic cascade leading to insufficient perfusion of the heart. The hypoxic injury results in permanent damage to heart tissue and, without successful intervention, frequently leads to CHF. In addition to drug-based therapies, major surgical procedures such as coronary artery bypass and medical devices providing mechanical augmentation for the ventricular myocardium are frequently administered, with significant risk of complications. Heart transplantation for post-MI patients with congestive heart failure is the most invasive procedure, considered extremely high-risk, and is limited by both organ availability and cost, making it unsustainable as a large scale solution. A highly successful and efficient therapy to replace damaged cardiac tissue and revitalize the cell-free fibrotic scar thereby preventing CHF has yet to be found.

Regeneration of the cardiac vasculature. The heart has the highest aerobic metabolic rate of any organ in the human body. The extensive nutrient and oxygen demand makes the maintenance and repair of heart vasculature essential both physiologically and post-injury. Endothelial cell damage plays an important role in cardiac pathologies such as atherosclerosis, thrombosis and hypertension, and physiological balance between endothelial cell loss and endothelial repair is crucial for reducing cardiovascular failure. Besides regrowth or repair of cardiomyocytes, efficient regenerative therapy following ischemic injury requires the repair of existing blood vessels as well as the formation of new blood vessels (neovascularization). While fully differentiated endothelial cells have a reduced capacity to regenerate vasculature, endothelial progenitor cells (EPCs) have been shown to be major contributors to angiogenesis and cardiac regeneration. Previous studies suggest that aging is associated with a reduced number and function endogenous EPCs. The combined effect of decreased EPC and BMSC numbers as well as function with age is a possible explanation for the limited cardiovascular regenerative potential in the elderly. An optimal cell type for cardiovascular repair should exert a significant beneficial effect on the vasculature in order to extend and prolong regeneration and reduce the risk of relapse and secondary heart failure. The vascular regenerative effect can derive from generating local, perfundable vasculature in situ and anastomose them with existing vessels and/or attracting blood vessel outgrowth from unaffected sites in the heart. Ultimately, the regenerated vasculature after an ischemic episode should resemble the features of healthy myocardium, both in terms of vascular density and blood vessel size in order to restore the heart's organized function as a whole.

MSC-based cell therapy for cardiovascular regeneration. Since the 1990s, many groups reported that implantation of healthy cells into the damaged myocardium can facilitate remodeling, enhance endogenous healing mechanisms and preserve ventricular structure and function in preclinical animal models of myocardial infarction (MI).The cellular and molecular mechanisms required for successful myocardial engraftment of transplanted cells have been described in detail elsewhere. Therapeutic cells need to survive and adapt in the microenvironment of the injured myocardium, promote the viability and proliferation of host cardiomyocytes, remodel the extracellular matrix (ECM) and activate resident and infiltrating progenitor cells to induce neoangiogenesis and cardiomyocyte turnover.

Mesenchymal stromal cells (MSCs), in particular bone marrow MSCs (BMSCs), have gained substantial interest in the context of tissue regeneration. MSCs can be isolated from many tissues, easily expanded, exhibit immunomodulatory effects, have properties of immunoprivilege, induce paracrine effects and they can be genetically modified in vitro. Extensive cardiac preclinical studies have demonstrated their safety and efficacy. Although the mechanism for the beneficial effects of cell transplantation remains unclear, ventricular function was improved in all studies.

Promising results from MSC-based pre-clinical studies has led to the initiation of greater than 20 NIH-registered clinical trials, which have mainly included autologous and allogeneic BMSCs. However, in sharp contrast to the improvements observed in animal models, the clinical trials using autologous cells produced only marginal benefits. Age-related limitations in the regenerative capacity of the implanted and endogenous progenitor cells are considered to be a major factor in the limited efficacy.

Human umbilical cord-derived perivascular cells. A young source of immuonoprivileged MSCs derived from the perivascular region of first trimester and term umbilical cord tissues is used, as described herein. First trimester human umbilical cord perivascular cells (FTM HUCPVCs) show pericyte-like characteristics, ability to differentiate into mesenchymal and non-mesenchymal lineages including cardiomyocyte-like cells. Superior multi-lineage differentiation, immunoprivileged and paracrine properties of FTM and also term HUCPVCs over BMSCs are exhibited. In this Example, it is examined whether FTM HUCPVCs have the ability to act at multiple levels of cardiovascular regeneration and their superior versatility could make these cells an optimal candidate for post-MI cell therapy.

Materials and Methods

All studies were performed with institutional research ethics board approval (REB No. 454-2011, Sunnybrook Research Institute; REB 29889, University of Toronto, Toronto, Canada). Established lines of FTM HUCPVCs and term HUCPVCs and a commercially available line of bone marrow MSCs (Lonza) were cultured in alpha-MEM (Gibco) supplemented with 10% FBS (Hyclone), and penicillin/streptomycin cocktail (Gibco), and passaged at 70-80% confluency. Rodent primary cardiomyocyte cultures were kept in DMEM-F12 containing 10% FBS (Hyclone) and penicillin/streptomycin cocktail (Gibco). MSC-monocyte co-cultures were kept in RPMI supplemented with 10% FBS (Hyclone) and penicillin/streptomycin. Cell cultures were kept in humidified incubators (37° C., 5% CO₂).

All animal procedures were conducted and reported according to ARRIVE guidelines and approved by the Animal Care Committee of the University Health Network (Toronto, Canada). All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health 1996). Primary rat cardiomyocyte cultures were prepared from rats sacrificed at p2-5. Ventricular myocardium was minced and agitated at 37° C. in 1.5% trypsin PBS solution. Primary cultures were treated with BrdU (16 h, 5 μM) and washed prior to addition of MSCs 24 hrs later. Dissociated MSCs were added to cardiomyocyte monolayers to achieve 5% MSC content in each well. Alternatively MSCs were labeled with viable, non-transferable fluorescent dye (CellTrackerGreen, Invitrogen 5 μM, 1 h) prior to transfer, in order to visualize integrating MSCs.

In vitro trans-membrane invasion assay. The extravasation capacity of MSCs in response to injured cardiomyocytes signals was evaluated using Corning Biocoat Transwell™ migration assays. Primary rat cardiomyocytes were plated in each well at high confluency. After cells attached, injury was applied by incisions (wound) or 24 h glucose/oxygen deprivation (ischemia), and FTM HUCPVC, term HUCPVC and BMSCs were plated on Matrigel™-coated membrane inserts separating human MSCs from rat cardiomyocytes. An endothelial cell invasion assay kit (BD #354141) was used to quantify trans basal membrane migration capacity of human MSCs. 30 k MSCs were plated on the Matrigel™-coated Transwell™ 3 μm pore plastic membrane inserts. Primary rat cardiomyocytes were seeded into 24 well tissue culture plates (200 k each). 24 h later, seeding media was replaced with FBS free D-MEM-F12 supplemented with 1% penicillin/streptomycin (assay media). Cardiomyocyte monolayers were injured by applying 6 radial incisions per well or incubating for 24 h in glucose free Ringers solution with air tight sealing (ischemia). Media was changed in ischemic wells to assay media before adding MSC-containing inserts. Inserts were incubated with cardiomyocyte wells for 24 h, stained with calcein-AM and scanned with a fluorescent plate reader (FilterMax F5 MolecularDevices) according to the manufacturer's description (BD). Cells were visualized by fluorescence microscopy (EVOS, LifeTechnologies).

In vitro wound healing assay. In order to assess the direct regenerative potential of MSCs on physically injured primary cardiomyocyte cultures, an in vitro wound healing assay was performed. A modified version of an in vitro scratch assay was applied. Briefly confluent, synchronously contracting rat primary cardiomyocyte culture was subjected to scratch incision using a sterile 20 gauge needle. FTM, term HUCPVCs and BMSCs were administered to the cardiomyocyte cultures 1 hour after the incisions were made. Unstained cell suspensions of MSCs were applied at the time of the incisions to observe their effect on wound closure. Pre-stained (CellTracker™ Green) FTM HUCPVCs were used to follow cell migration in the wounded cardiomyocyte culture. Cardiomyocytes were counterstained with CellTracker™Orange CMRA. Both intact and wounded fields were imaged for 48 h with fluorescence microscopy (EVOS, LifeTechnologies) to record changes in localization of CTG positive cells. After 48 h real-time fluorescence and bright field microscopy video recordings were acquired (EVOS, LifeTechnologies, Epiphan Systems) to assess for contractions of primary cardiomyocyte fields and the distribution of labelled cells in the vicinity of the wounds. Wound healing of primary cardiomyocyte monolayers co-cultured with MSCs was followed for 1 week by phase contrast and fluorescence microscopy.

Cell content transfer between HUCPVCs and cardiomyocytes. To assess mechanisms of direct cell-cell communication, HUCPVCs were loaded with transferable cytoplasmic fluorophore CellTracker™Orange CMTMR and rat cardiomyocytes were labelled with non-transferrable CellTracker™Green: CTG prior to establishing direct co-cultures. The mitochondrial fraction of mammalian cells was labelled with Rhodamine123, that is selectively accumulated by live mitochondria with high membrane potential. To assess for mitochondrial transfer, undifferentiated FTM HUCPVCs were incubated with non-transferable cytoplasmic fluorescent dye (CellTracker™Green) and live mitochondrial dye Rhodamine123. A single cell suspension of double-stained FTM HUCPVCs was added to unlabelled primary rat cardiomyocyte cultures and allowed to integrate for 24. Transfer of mitochondrial fluorophore was assessed by fluorescence microscopy for 72 hours.

Flow cytometry and FACS. For FC and FACS, cell cultures were dissociated with 0.25% Trypsin/EDTA solution (3 min 37° C.). Cell suspensions were incubated with fluorophore conjugated primary antibodies according to provider's description (1:40, 30 min 4° C.). FC analysis was performed using either analogue (FacsCalibur, BD; Create Fertility Centre, Toronto) or digital (LSR II., Canto II., BD; UHN SickKids Flow Cytometry Facility, Toronto) analytical cytometers. FACS was performed using digital cell sorters (MoFlo Astrios, Aria II., UHN SickKids Flow Cytometry Facility, Toronto) and sorted cells were re-plated within 1 hour after the procedure. MSC treated rat hearts were processed for flow cytometry analysis to evaluate immunologically relevant cell surface molecules HLA-A and HAL-G levels of the engrafted human cells 2 weeks after administration.

Rat aortic ring assay. Aortic tissues were isolated from female rats of reproductive age. After sectioning and embedding in an extracellular matrix extract (Matrigel™), sprouting endothelial networks were monitored and imaged until closed loops and structured networks were established. Human MSCs expanded in serum-containing conditions (alpha-MEM, 10% FBS) up to passage 4-6 and labeled with CellTrackerGreen™ were seeded onto Matrigel™-embedded aortic endothelium originated networks (10 k MSCs per mm aortic tissue section). After 24 hours of incubation, bright field and fluorescent images were taken to document overall network development, MSC localization and physical interactions between MSCs and endothelial cells. Phase contrast images consistent for size of fields of sight were taken of endothelial networks to measure radial growth of endothelial network and total number of closed loops for up to 7 days. In aortic ring originated endothelial networks uniform quadrants (n=12) circumscribing areas of developed tube networks were defined and number of closed loops were compared between treatment groups (Image J).

Immunocompromised rat model of MI. Surgeries were performed on anesthetized immunocompromised rats (Foxn1mu homozygous) by certified animal surgeons (University Health Network, AUP #1059.27). Myocardial infarction was induced by permanent left coronary artery ligation. One week after MI, cells (3×106) were administered in 3 injections directly into the anterior myocardium of the left ventricle, around the ischemic region. Echocardiography was performed using ACUSON SEQUOIA C256 System (SIEMENS Medical Solutions USA, Inc; California, USA) 2 weeks after cell treatment to assess cardiac function.

Immunocompromised mouse model of MI. Nude (NOD scid) mice were subjected to coronary artery ligation under isoflurane anaesthesia and within 24 h they were injected with human MSC suspensions or media as control. Injections were administered into the apical myocardium of the left ventricule (10⁶ cells each). Echocardiography was following cardiac output for up to 6 months.

Histological evaluation of rat heart tissue. Rat hearts were ectomized at 2 weeks after MSC administration and processed for FC, basic histological evaluation and immunohistochemistry (IHC). For FC, cells were harvested from the anterior myocardium of the left ventricle using trypsin/collagenase treatment. HLA-A and HLA-G levels were analyzed in the TRA-1-85high fraction by flow cytometry, to evaluate immunologically relevant cell surface molecules in the engrafted human cells 2 weeks after administration.

For basic histological analyses, rat hearts from each experimental group were perfused and fixed with 10% formalin and embedded in paraffin for sectioning. Masson's trichrome staining was performed on heart sections at three levels of the scarred ventricles to quantify scar tissue formation. Vascular formation and density was assessed on deparaffinized tissue sections using endothelial membrane specific fluorescent isolectin (IB4, LifeTechnologies, 100× dilution in PBS).

Fluorescent in situ hybridisation (FISH) and qPCR for human genomic DNA tracing. Human cell retention was assessed qualitatively and quantitatively in MSC treated heart tissues applying previously reported primers for Alull human retrotransposon (10⁶ copies per genome). Alull FISH probe (ALU, green) was applied for human cell tracing in the ischemic regions of the rat myocardium. Human umbilical cord section (human cord) was used for positive and untreated rat heart (rat heart no tx) as negative control. Genomic qPCR was performed. FITC conjugated oligos of the same sequences were applied for FISH (ACGT Corp. Toronto Canada). Briefly, deparaffinised tissue sections were subjected to epitope retrieval (pH6), permeabilization (triton-X100, 0.1%) and incubated with fluorophore conjugated oligos (200 nM) in hybridisation buffer (20 mM Tris pH7.4, formamide, blocking solution (Roche)).

Statistical Analysis. ANOVA followed by Multiple t tests (grouped analysis) were performed to detect and verify statistically significant differences (α<0.01) between cell lines using Prism software (Graph Pad). p value was calculated using a one-way ANOVA and Tukey's Post test.

Sample size for flow cytometry (FC) measurements were: FTM n=9, term n=9, BMSC n=6 independent experiments using 2 lines per cell type; for vascular IHC: n≥20 random fields of sight were taken of 3 animals per experimental group. Aortic ring assay: N=3 rings per treatment group, n=4 fields of sight. Echocardiography n=6 animals per treatment group. Alu genomic qPCR: 5 samples per experimental group were tested.

Results

It was found that FTM HUCPVCs actively migrate towards injured cardiomyocytes through basal membrane-like structures. Quantification of fluorescently-labeled MSCs in the trans-membrane invasion assay revealed that cardiomyocyte injury significantly increased active migration of 2 independent lines of FTM HUCPVCs in comparison to non-injured controls (p<0.01). This effect was not observed for term HUCPVCs. The transmembrane migration of BMSCs was significantly inhibited by cardiomyocyte injury (p<0.01).

Further, it was found that FTM HUCPVCs promote wound healing and reconnect cardiomyocytes after injury in vitro. In order to assess the direct regenerative potential of MSCs on physically injured primary cardiomyocyte cultures, an in vitro wound healing assay was performed. Straight incisions closed after 1 week in both FTM and term HUCPVC-containing co-cultures, while BMSC-containing primary cardiomyocyte cultures still contained uncovered, open wounds after 1 week. Closer inspection of the closed wounds (10×) revealed that while term HUCPVC-treated incisions were bridged by the protrusions of the cells on the edges of the wounded cardiomyocyte sheets, FTM HUCPVC containing co-cultures had a high number of cell bodies filling the grooves between cardiomyocyte sheets with different morphology than those of the original primary cardiac culture. In order to clarify the contribution of FTM HUCPVCs to the observed wound healing effect, cells were pre-stained with viable fluorescent dye (CellTracker™ Green, CTG) prior to administration onto injured cardiomyocyte monolayers. CTG-loaded FTM HUCPVCs were initially randomly distributed shortly after plating (3 h). Over time CTG positive cells were found in increasing abundance in the vicinity of injured cardiomyocytes (16 h) and ultimately covered the wounds (48 h). Employing fluorescently labelled co-cultures, high magnification (200×) fluorescent microscopy images showed that instead of cells from the original primary culture (CM, red), FTM HUCPVCs (FTM, green) almost exclusively served as the building blocks to reconnect separated cardiomyocyte fields. Furthermore, it was evident that HUCPVCs initially (24 h) connected with, and covered, the wounded edges instead of attaching to cell-free surfaces. In time (48 h) HUCPVCs appeared to be expanded and reached the opposite side of the wound. Live imaging of injured cardiomyocyte co-cultures shows that pre-stained cardiomyocyte fields separated by incisions lose their synchronised contraction. Following administration of FTM HUCPVCs, the injured cultures reached critical abundance at the injury site, and they gradually connected the opposing sides of the wound. Following the reconnection, contraction waves begin to pass through the previously isolated fields.

It was found that FTM HUCPVCs transfer cytoplasmic content into rat cardiomyocytes in vitro. Functional integration of the implanted cells into the myocardium requires the establishment of functional interactions with cardiomyocytes. The non-transferable nature of CTG was confirmed by staining co-cultures for human nucleus specific marker (HuNu). CTO stain appeared in rat cardiomyocytes after 3 days of co-culture. Furthermore, only rat cardiomyocytes in direct contact with CTO-containing human cells showed dye uptake, suggesting that trans-membrane junctions may have formed.

It was observed that FTM HUCPVCs transfer mitochondrial content into rat cardiomyocytes in vitro. The observation regarding the transfer of cytoplasmic content from FTM HUCPVCs into rat primary cardiomyocytes in co-cultures raised the question of whether the transfer of such compounds has any physiological relevance. After 72 hours of co-culture, mitochondrial-associated signals derived from FTM HUCPVCs appeared in the cardiomyocytes (CM) that were in direct contact with integrated FTM HUCPVCs, suggesting direct cell-to-cell transfer of mitochondrial content.

FTM HUCPVCs were seen to have higher connexin43 expression than term HUCPVCs and BMSCs when co-cultured with rat cardiomyocytes. Direct cell-to-cell interaction and communication requires intercellular membrane channel complexes between connecting cells. Primary cardiomyocyte cultures were isolated from ventricular regions of rat pup hearts as described above. In primary culture, cardiomyocytes show strong staining for connexin43 (cx43). When separated by the non-cardiomyocyte cells found in primary culture (likely fibroblasts) cardiomyocytes demonstrated very few cx43 positive bridges between them. In contrast, co-cultures of first trimester (FTM) HUCPVCs with rat cardiomyocytes showed distinct puncta that appeared to connect the cardiomyocyte cells. Under the same conditions, term HUCPVC co-cultures contained similar puncta, but to a lesser degree. Whereas with BMSC co-cultures puncta were not observed. Flow cytometric analysis was performed on co-cultures to quantify the proportion of cx43-positive human cells (TRA-1-85+, cx43+ double positive cells). The three types of human MSCs from co-cultures (1 week) showed different ratios of positivity for cx43. When comparing the extent of staining, FTM HUCPVCs showed the highest ratio of cx43+ cells (30%) when compared to term HUCPVCs (21%) and BMSCs (12%). The amount and distribution of cx43 signal suggests that gap junctions are responsible for the observed cellular content transfer between FTM HUCPVCs and cardiomyocytes, and the resynchronisation of separated cardiomyocyte fields.

FTM HUCPVCs were seen to integrate with developing endothelial networks and results in higher network growth when compared to other MSCs in rat aortic ring assay. Pre-stained (CTG, CellTrackerGreen™) suspensions of human MSCs were administrated to developing endothelial networks of ex-vivo cultured rat aortic rings in order to observe cell integration and effect on network growth. CTG-positive cells were imaged within the network 24 h after cell administration. FTM HUCPVCs appeared to preferentially associate with the peripheral, developing areas of the endothelial networks and displayed an elongated morphology. In contrast, term HUCPVCs and BMSCs showed little site preference and also localized within areas with minor tube formation.

Total network growth was calculated as the distance between the first proximal closed network loops near the aortic ring and the most distal closed loop. The mean radial size of networks co-cultured with MSCs were as follows: FTM HUCPVCs 2.9±0.3 mm, term HUCPVCs 2.2±0.5 mm, BMSCs 1.7±0.3 mm (n=3 for each condition). Untreated rings developed endothelial networks with a mean radius of 2.4±0.5 mm. Statistical comparison of MSC treatment groups showed that FTM HUCPVCs resulted in significantly increased network growth compared to term HUCPVC (p≤0.05) and BMSC co-cultures (p≤0.01). Untreated endothelial cultures expressed significantly greater network growth when compared to BMSC co-cultures (p≤0.05).

Regarding network structure analyses, the average number of total closed loops in FTM HUCPVC-treated aortic ring networks (70±43) was significantly higher than term HUCPVCs (29±12) and BMSCs (24±2) (p≤0.05). Untreated rings (79±14) displayed similar loop numbers compared to FTM HUCPVC co-culture conditions, but was significantly higher than those of either term HUCPVC or BMSC treated networks (p≤0.01).

Implantation of FTM HUCPVCs in a rat model with MI leads to diminished scar tissue formation, greater cell retention, increased vascularisation and significant improvements in cardiac function when compared to term HUCPVCs or BMSCs.

Two weeks after cell injection, hearts were sectioned and tissue was analyzed for fibrotic tissue content at the level of ischemic injury. Mean scar tissue mass proportion to normal tissue in FTM HUCPVC, term HUCPVC and BMSC-treated hearts was 15±9%, 18±10%, and 36±9% respectively. Fibrotic tissue was significantly decreased in FTM HUCPVC when compared to BMSC-treated hearts (p<0.05).

Fluorescent microscopy images showed a higher frequency of Alullb+ (human) nuclei in the scar tissue regions of the FTM HUCPVC-treated post-MI rat hearts compared to term HUCPVC- and BMSC-treated samples. Human cell content of ischemic rat hearts was quantified by genomic qPCR for Alullb and correlated to scar tissue content. Hearts treated with FTM HUCPVCs after MI showed significantly higher human nuclear DNA content when compared to term HUCPVCs and BMSC-treated hearts post-MI (p<0.05) and showed an inverse relationship with the amount of fibrotic tissue.

HUCPVCs show low immunogenicity when isolated from rat heart 2 weeks after administration.

Two weeks after injection 55% and 65% of the TRA-1-85high (human) cells isolated from FTM and term HUCPVC-treated rat hearts were found to express HLA-A, while 25% and 20% expressed of FTM HUCPVCs and term HUCPVCs expressed HLA-G, respectively. This indicates low immunogenicity even after 2 weeks in the myocardial niche. The number of TRA-1-85high (human) cells within cardiac tissue after treatment with BMSCs was below the amount required for immunophenotypical analysis (data not shown).

It was found that FTM HUCPVC treatment improves vascularisation of the ischemic myocardium after MI. Fluorescent isolectin (IB4) was utilized as an endothelial-specific probe in myocardial tissue sections to reveal blood vessel abundance and distribution in the ischemic heart muscle. As described above, fluorescent microscopy images were quantified for number and size of IB4-positive entities (capillaries). Vascular density of the ischemic myocardium was significantly higher (p<0.01) in FTM and term HUCPVC treated hearts compared to untreated (MI only). BMSC treatment did not result in a significantly increased capillary density. Median area of blood vessel cross-sections was the highest among the 3 cell types after FTM HUCPVC treatment and was significantly higher (p<0.01) than in BMSC treated hearts, but not different than untreated MI vessels.

It was found that FTM HUCPVC treatment leads to significant functional improvement after MI. Cardiac output of the animals (Foxrun rats) in every treatment group was assessed by echocardiograpy. Functional improvements in cardiac output at 2 weeks post-intramyocardial cell implantation were observed in rat models of MI with all three cell types when compared to those with media injection. However, left ventricular internal dimension (LViDs) was significantly lower in animals treated with FTM HUCPVCs, but not the other 2 cell types (p<0.01 and fractional shortening (% FS) was significantly higher in FTM HUCPVC-treated animals, compared to untreated (p<0.01).

FTM HUCPVC treatment was found to induce sustained functional improvements in a mouse model of MI for up to 6 months. In an in vivo study, FTM and term HUCPVCs were implanted acutely into an immunocompromised (NOD scid) mouse model of MI. Echocardiography showed that FTM HUCPVCs have an increased ability to restore heart function following MI when compared to term HUCPVCs. Ejection fraction and fractional shortening was significantly higher, while left ventricular internal diameter was significantly lower in FTM HUCPVC-treated animals when compared to those treated with term HUCPVCs, respectively. These effects are apparent as early as 2 weeks following MI and are sustained for up to 6 months. Endpoint histology analysis on post-MI mouse hearts six weeks after administration showed that scar area is significantly lower and scar thickness is significantly higher in HUCPVC-treated hearts compared to media controls (p=0.05). Further analysis of FTM and term HUCPVC-treated experimental groups showed that these physical improvements are significantly increased in FTM HUCPVCs when compared to term HUCPVCs (p<0.05). The functional improvement of HUCPVC-treated post-MI mouse hearts was sustained up to 6 months after administration with FTM HUCPVCs maintaining higher ejection fraction and fractional shortening and lower left ventricular dimension than term HUCPVCs (p<0.05). This sustained effect can be a crucial advantage in large animal models of longer life-span.

Discussion

The ultimate goals of a successful regenerative cell therapy for congestive heart failure is clear from a functional and histological aspect. However, the underlying cellular mechanisms required to achieve this are multifaceted and complex in nature. While a candidate cell type may have beneficial effects on one or more aspects of the regenerative process (eg proliferation, immunomodulation, and remodelling), only cell types capable of fulfilling all aspects of tissue repair would be optimal for cell therapy. Pericytes are localized around blood vessels in every tissue of the human body and have a crucial role in blood vessel formation and support, as well as controlling transport mechanisms between the tissue constituent cells and their circulation. The regenerative importance of pericytes and pericyte-derived MSCs is becoming increasingly evident. Term HUCPVCs could contribute to neoangiogenesis, endogenous cell recruitment, and immunomodulation. Although other groups have shown that term HUCPVCs displayed induced expression of cardiac-related marker expression in vitro, the same studies did not find a significant change in scar tissue formation or cardiac function in an in vivo MI model, suggesting the absence of significant tissue remodelling using these cells. Cell therapy applied either locally or systemically requires that the administered cells home preferentially to sites of injury; thus a successful cell therapy candidate should possess or develop such ability. As shown herein, assessment of in vitro cell invasion demonstrated that FTM HUCPVCs are attracted towards injured cardiomyocytes, and actively migrate through basal membrane-like barriers. Term HUCPVCs did not display this capacity and BMSC migration was inhibited by the proximity of injured cardiac cells.

After cardiac injury, the disruption of functional intercellular connections due to cell loss or separation by pathological ECM often leads to akinetic or arrhythmic tissue. Functional restoration of the myocardium necessitates the reestablishment of cell-cell connections. The observed appearance of cx43-positive puncta on the FTM HUCPVCs between rat cardiomyocytes in co-cultures and the transfer of gap junction permeable fluorophores, suggest functional intercellular connections. Induction of the major gap junction protein, connexin43, in co-cultures of stem cells and cardiomyocytes may improve intercellular signaling and progressively increase conductivity. The reconnection of separated cardiomyocyte fields as seen herein suggests that electrophysiological connections are established between FTM HUCPVCs and rat cardiomyocytes. In order to avoid fibrillations and arrhythmia in vivo, implanted cells are required to inherently possess or develop such features. From the current data, it is apparent that FTM HUCPVCs could serve as supporting cells in vivo after MI, in part by helping to restore cardiac tissue conductance.

Healthy heart tissue has the highest oxygen consumption compared to any other organ in the human body, both in resting state (8 ml O₂/min per 100 g) and during exercise (70 ml O₂/min per 100 g). This intense aerobic metabolism requires a high number of functional mitochondria (˜5000 per cardiomyocyte) which decreases with age in mammals. Mitochondrial impairment in the heart muscle is a component of many cardiac pathologies and is often a therapeutic target. It has been reported that mesenchymal stromal cells have the potential for cell to cell support and regeneration of mitochondria and in particular, MSCs can regenerate the mitochondria of ischemic cardiomyoblasts. In this Example, it was observed an intense transfer of mitochondria-associated fluorophores from FTM HUCPVCs into primary cardiomyocytes in co-cultures, within 4 days of co-culture. This most likely represents intercellular transfer of mitochondria from FTM HUCPVCs into cardiomyocytes. A less likely, but possible explanation is the transfer of FTM HUCPVC's mitochondrial content through gap junctions, which is subsequently taken up and accumulated in the recipient cardiomyocyte's mitochondrial compartment. This may be another mechanism by which FTM HUCPVCs could potentially support cardiac tissue repair.

In the rat aortic ring assay, FTM HUCPVCs showed the highest interaction with endothelial networks and supported the development of endothelial networks more than term HUCPVCs and BMSCs. The radial growth of the in vitro Matrigel™-embedded rat aortic ring endothelial network appeared to be the combined result of endothelial cell proliferation and elongation and the capability of the tubular network to remodel the surrounding extracellular matrix. Conditions resulting in higher radial growth of endothelial cell populations suggest a higher tissue penetration rate when translated to in vivo models.

Besides the rate of radial network growth, the network structure of the emerging neo-vasculature must be optimal for efficient tissue regeneration. A higher number of closed loops in a functional capillary network would result in a higher perfusion rate, within a given tissue area. Higher radial growth combined with higher number of closed loops in the FTM HUCPVC treatment group in vitro, could translate into higher surface coverage of an endothelial network in vivo. This is consistent with the in vivo observation that vasculature in the scar tissue of FTM HUCPVC treated ischemic hearts significantly improved compared to an untreated animal after MI. Importantly, FTM HUCPVCs were found in the scar tissue in the highest amount compared to term HUCPVCs and BMSCs, and the human cell abundance showed a reverse correlation with the extent of fibrotic tissue. Assuming that during the 1 week between MI induction and cell implantation the majority of vascular degradation has run its course in the ischemic myocardium, it also has to be considered that higher vascular density compared to untreated hearts can be, at least in part, due to neovascularization. This neovasculature must also be of perfundable diameter, thus only the combined effect of increased number and an increased, or maintained, blood vessel size would be able to achieve a functional vascular regenerative effect. From the observations made in this Example on the three cell types examined, it appears that FTM HUCPVCs would likely be the best candidate to accomplish this type of vascular regeneration and provide adequate reperfusion in a clinical setting. It was notable to observe that the highly promising results observed in vitro, together with the histological changes observed in vivo that FTM HUCPVCs—as opposed to term HUCPVCs and BMSCs—translated into a significant functional regenerative effect on post-MI hearts 2 weeks after administration. Furthermore, HUCPVCs isolated from rat hearts also showed a favourable immunological phenotype, suggesting that in vivo integration does not compromise their immune-privileged nature, and may augment their retention for a longer period of time. In an immunocompromised mouse model of MI, the FTM HUCPVCs' beneficial effect was sustained for up to 6 months.

Extended retention of FTM HUCPVCs in the infarcted heart can be sufficient for their superior regenerative mechanisms to act and have provide sustained beneficial effects necessary in pre-clinical and clinical scenarios alike.

In summary, FTM HUCPVCs have in vitro properties consistent with a cell type that could be effective for cell therapy post MI. These properties were enhanced when compared to term HUCPVCs or BMSCs. In concert with this, they exhibited sustained and superior functional improvement in rodent in vivo models, as compared to the other older MSC sources tested. Therefore, FTM HUCPVCs are a promising new candidate for regenerative cellular therapy after MI.

In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that these specific details are not required in order to practice the technology.

The above-described embodiments are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art without departing from the scope, which is defined solely by the claims appended hereto. All documents referred to herein are incorporated by reference. 

The invention claimed is:
 1. A method of isolating pluripotent human umbilical cord perivascular cells for treating cardiovascular injury or heart disease, wherein the isolated perivascular cells differentiate into cardiomyocytes expressing cTnI, wherein the pluripotent human umbilical cord perivascular cells are obtained by: collecting an umbilical cord from fetal tissue obtained at less than 13 weeks of gestation, by collecting fetal placenta tissue by surgical aspiration; and separating the umbilical cord from the fetal placenta tissue; treating the umbilical cord to obtain isolated perivascular cells, by: washing the umbilical cord with PBS, cutting the umbilical cord, comprising an artery and two vessels, into smaller cut pieces, treating the cut pieces with collagenase to obtain isolated perivascular cells, and washing the isolated perivascular cells with PBS; incubating the isolated perivascular cells, by suspending the isolated perivascular cells in a maintenance medium comprising a-MEM, penicillin-streptomycin, and amphotericin; maintaining the isolated perivascular cells by: washing the isolated perivascular cells with PBS; adding trypsin-EDTA; harvesting the isolated perivascular cells into a tube containing maintenance medium; separating the isolated perivascular cells from the maintenance medium; mixing the isolated perivascular cells with new maintenance medium; diluting the new maintenance medium containing the isolated perivascular cells with additional maintenance medium to obtain a diluted maintenance medium containing the isolated perivascular cells; maintaining the diluted maintenance medium containing the isolated perivascular cells under appropriate growth conditions of 37° C., 5% CO2 in a maintenance medium, and changing the maintenance medium every 3-7 days; and suspending the maintained cells, wherein prior to cutting the umbilical cord into smaller cut pieces, the umbilical cord is from 0.5-2.0 cm in length and comprises an artery; wherein the isolated perivascular cells express one or more transcription factor associated with undifferentiated stem cells; and the transcription factor is OCT-4, SOX-2, or Nanog.
 2. The method according to claim 1, wherein maintaining the isolated perivascular cells additionally comprises: freezing the isolated perivascular cells; and thawing and restoring the isolated perivascular cells to viability.
 3. The method according to claim 1, wherein the collagenase is Type I at 1 mg/mL.
 4. The method according to claim 2, wherein freezing the isolated perivascular cells comprises: washing the isolated perivascular cells with PBS; adding trypsin-EDTA; harvesting the isolated perivascular cells into a tube containing maintenance medium; separating the isolated perivascular cells from the maintenance medium; cooling the isolated perivascular cells to 4° C.; mixing the isolated perivascular cells with a freezing medium at 4° C., said freezing medium comprising DMSO; transferring the freezing medium containing the isolated perivascular cells to vials pre-chilled to −70° C.; storing the vials at −70° C. for 24 h; and storing the vials in liquid nitrogen.
 5. The method according to claim 2, wherein thawing and restoring the isolated perivascular cells comprises: warming the vials 37° C.; separating the isolated perivascular cells from the freezing medium; mixing the isolated perivascular cells with maintenance medium; maintaining the maintenance medium containing the isolated perivascular cells under appropriate growth conditions of 37° C., 5% CO₂, for 24 hours; replacing the maintenance medium with new maintenance medium; and maintaining the new maintenance medium containing the material under appropriate growth conditions, said conditions being 37° C., 5% CO₂ and changing the new maintenance medium every 3-7 days.
 6. The method according to claim 1, wherein maintaining the isolated perivascular cells further comprises: (i) aspirating the medium from the cells when the density of cells exceeds about 70% of the surface of a culture dish or flask in which the cells are maintained, (ii) washing the cells with PBS; (iii) adding trypsin-EDTA to cover the cells until the cells lift off of the dish or flask; and (iv) resuspending the cells in maintenance medium. 